Effect of lipopolysaccharide on osteoclasts formation and bone resorption function and its mechanism_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZENG Li ¹ , XU Yongming ² ,  XING Gengyan ²

1. General Hospital of Chinese People’s Armed Police Forces of Jinzhou Medical University Postgraduate Training Base, Beijing, 100039, P.R.China;
2. Department of Orthopedics, General Hospital of Chinese People’s Armed Police Forces, Beijing, 100039, P.R.China;

Corresponding author：

XING Gengyan, Email: xgy1350138@foxmail.com

Keywords：

Osteoclasts; bone marrow-derived macrophages; lipopolysaccharide; Connexin43; mouse

DOI：

10.7507/1002-1892.201712044

Video：

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ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area.ResultsThe flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.

Citation： ZENG Li, XU Yongming, XING Gengyan. Effect of lipopolysaccharide on osteoclasts formation and bone resorption function and its mechanism. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(5): 568-574. doi: 10.7507/1002-1892.201712044 Copy

1.	Karsenty G. The complexities of skeletal biology. Nature, 2003, 423(6937): 316-318.
2.	Zeng XZ, He LG, Wang S, et al. Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression. Acta Pharmacol Sin, 2016, 37(2): 255-263.
3.	Qu X, Zhai Z, Liu X, et al. Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades. Biochem Biophys Res Commun, 2014, 443(2): 658-665.
4.	Zhao N, Tsuda H, Murofushi T, et al. Chaetocin inhibits RANKL-induced osteoclast differentiation through reduction of Blimp1 in Raw264.7 cells. Life Sci, 2015, 143: 1-7.
5.	Choe JY, Kim SK. Melittin inhibits osteoclast formation through the downregulation of the RANKL-RANK signaling pathway and the inhibition of interleukin-1β in murine macrophages. Int J Mol Med, 2017, 39(3): 539-548.
6.	Ye S, Fowler TW, Pavlos NJ, et al. LIS1 regulates osteoclast formation and function through its interactions with dynein/dynactin and Plekhm1. PLoS One, 2011, 6(11): e27285.
7.	Strålberg F, Kassem A, Kasprzyowski F, et al. Inhibition of lipopolysaccharide-induced osteoclast formation and bone resorption in vitro and in vivo by cysteine proteinase inhibitors. J Leukoc Biol, 2017, 101(5): 1233-1243.
8.	Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A, 1998, 95(7): 3597-3602.
9.	Boyce BF. Advances in the regulation of osteoclasts and osteoclast functions. J Dent Res, 2013, 92(10): 860-867.
10.	Chung YH, Chang EJ, Kim SJ, et al. Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts. J Periodontal Res, 2006, 41(4): 288-296.
11.	Islam S, Hassan F, Tumurkhuu G, et al. Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells. Biochem Biophys Res Commun, 2007, 360(2): 346-351.
12.	Zhai ZJ, Li HW, Liu GW, et al. Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo. Br J Pharmacol, 2014, 171(3): 663-675.
13.	Qin A, Cheng TS, Lin Z, et al. Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption. PLoS One, 2012, 7(4): e34132.
14.	Zou W, Izawa T, Zhu T, et al. Talin1 and Rap1 are critical for osteoclast function. Mol Cell Biol, 2013, 33(4): 830-844.
15.	Ilvesaro J, Väänänen K,Tuukkanen J. Bone-resorbing osteoclasts contain gap-junctional connexin-43. J Bone Miner Res, 2000, 15(5): 919-926.
16.	Hobolt-Pedersen AS, Delaissé JM, Soe L. Osteoclast fusion is based on heterogeneity between fusion partners. Calcif Tissue Int, 2014, 95(1): 73-82.
17.	Kang JH, Sim JS, Zheng T, et al. F4/80 inhibits osteoclast differentiation via downregulation of nuclear factor of activated T cells, cytoplasmic 1. Arch Pharm Res, 2017, 40(4): 492-499.
18.	Lee K, Chung YH, Ahn H, et al. Selective regulation of MAPK signaling mediates RANKL-dependent osteoclast differentiation. Int J Biol Sci, 2016, 12(2): 235-245.
19.	Kylmäoja E, Kokkonen H, Kauppinen K, et al. Osteoclastogenesis is influenced by modulation of gap junctional communication with antiarrhythmic peptides. Calcif Tissue Int, 2013, 92(3): 270-281.
20.	Shi C, Zhang H, Louie K, et al. BMP signaling mediated by BMPR1A in osteoclasts negatively regulates osteoblast mineralization through suppression of Cx43. J Cell Biochem, 2017, 118(3): 605-614.
21.	Schilling AF, Filke S, Lange T, et al. Gap junctional communication in human osteoclasts in vitro and in vivo. J Cell Mol Med, 2008, 12(6A): 2497-2504.
22.	Xing Q, de Vos P, Faas MM, et al. LPS promotes pre-osteoclast activity by up-regulating CXCR4 via TLR-4. J Dent Res, 2011, 90(2): 157-162.

1. Karsenty G. The complexities of skeletal biology. Nature, 2003, 423(6937): 316-318.
2. Zeng XZ, He LG, Wang S, et al. Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression. Acta Pharmacol Sin, 2016, 37(2): 255-263.
3. Qu X, Zhai Z, Liu X, et al. Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades. Biochem Biophys Res Commun, 2014, 443(2): 658-665.
4. Zhao N, Tsuda H, Murofushi T, et al. Chaetocin inhibits RANKL-induced osteoclast differentiation through reduction of Blimp1 in Raw264.7 cells. Life Sci, 2015, 143: 1-7.
5. Choe JY, Kim SK. Melittin inhibits osteoclast formation through the downregulation of the RANKL-RANK signaling pathway and the inhibition of interleukin-1β in murine macrophages. Int J Mol Med, 2017, 39(3): 539-548.
6. Ye S, Fowler TW, Pavlos NJ, et al. LIS1 regulates osteoclast formation and function through its interactions with dynein/dynactin and Plekhm1. PLoS One, 2011, 6(11): e27285.
7. Strålberg F, Kassem A, Kasprzyowski F, et al. Inhibition of lipopolysaccharide-induced osteoclast formation and bone resorption in vitro and in vivo by cysteine proteinase inhibitors. J Leukoc Biol, 2017, 101(5): 1233-1243.
8. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A, 1998, 95(7): 3597-3602.
9. Boyce BF. Advances in the regulation of osteoclasts and osteoclast functions. J Dent Res, 2013, 92(10): 860-867.
10. Chung YH, Chang EJ, Kim SJ, et al. Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts. J Periodontal Res, 2006, 41(4): 288-296.
11. Islam S, Hassan F, Tumurkhuu G, et al. Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells. Biochem Biophys Res Commun, 2007, 360(2): 346-351.
12. Zhai ZJ, Li HW, Liu GW, et al. Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo. Br J Pharmacol, 2014, 171(3): 663-675.
13. Qin A, Cheng TS, Lin Z, et al. Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption. PLoS One, 2012, 7(4): e34132.
14. Zou W, Izawa T, Zhu T, et al. Talin1 and Rap1 are critical for osteoclast function. Mol Cell Biol, 2013, 33(4): 830-844.
15. Ilvesaro J, Väänänen K,Tuukkanen J. Bone-resorbing osteoclasts contain gap-junctional connexin-43. J Bone Miner Res, 2000, 15(5): 919-926.
16. Hobolt-Pedersen AS, Delaissé JM, Soe L. Osteoclast fusion is based on heterogeneity between fusion partners. Calcif Tissue Int, 2014, 95(1): 73-82.
17. Kang JH, Sim JS, Zheng T, et al. F4/80 inhibits osteoclast differentiation via downregulation of nuclear factor of activated T cells, cytoplasmic 1. Arch Pharm Res, 2017, 40(4): 492-499.
18. Lee K, Chung YH, Ahn H, et al. Selective regulation of MAPK signaling mediates RANKL-dependent osteoclast differentiation. Int J Biol Sci, 2016, 12(2): 235-245.
19. Kylmäoja E, Kokkonen H, Kauppinen K, et al. Osteoclastogenesis is influenced by modulation of gap junctional communication with antiarrhythmic peptides. Calcif Tissue Int, 2013, 92(3): 270-281.
20. Shi C, Zhang H, Louie K, et al. BMP signaling mediated by BMPR1A in osteoclasts negatively regulates osteoblast mineralization through suppression of Cx43. J Cell Biochem, 2017, 118(3): 605-614.
21. Schilling AF, Filke S, Lange T, et al. Gap junctional communication in human osteoclasts in vitro and in vivo. J Cell Mol Med, 2008, 12(6A): 2497-2504.
22. Xing Q, de Vos P, Faas MM, et al. LPS promotes pre-osteoclast activity by up-regulating CXCR4 via TLR-4. J Dent Res, 2011, 90(2): 157-162.

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Effect of lipopolysaccharide on osteoclasts formation and bone resorption function and its mechanism

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