STUDY ON MOLECULAR MECHANISM OF OSTEOCLAST DIFFERENTIATION INDUCED BY STAPHYLOCOCCAL PEPTIDOGLYCAN_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

RENLirong ¹ ,  XUYongqing ² , WANGHai ² , HEXiaoqing ² , SONGMuguo ² , CHENXueqiu ¹

1. Kunming Medical University, Kunming Yunnan, 650504, P. R. China;
2. Department of Orthopedics, Kunming General Hospital of Chengdu Military Region;

Corresponding author：

XUYongqing, Email: xuyongqingkm@163.net

Keywords：

Staphylococcal peptidoglycan; Osteoclast differentiation; Nuclear factor κB; Osteomyelitis; Molecular mechanism

DOI：

10.7507/1002-1892.20160254

Video：

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Objective To investigate the molecular mechanism of osteoclast differentiation induced by Staphylococcal peptidoglycan (PGN-sa). Methods Raw264.7 cells were stimulated with PGN-sa and with PGN-sa+SC75741[a potent inhibitor of nuclear factor κB (NF-κB) activation] in a concentration of 200 ng/mL. The protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was tested at 0, 1, 2, and 3 days; the proteins related to osteoclast differentiation of extracellular regulated protein kinases (ERK), p38, c-Jun N-terminal kinase (JNK), NF-κB, inhibitor of NF-κB (IκB-α), Akt, and the phosphorylation forms of p38, ERK, JNK, Akt, NF-κB were measured at 0, 5, 10, 20, 40, and 60 minutes by Western blot. In addition, Raw264.7 cells were stimulated with PGN-sa in the concentrations of 100 ng/mL (group A), 200 ng/mL (group B), 400 ng/mL (group C), and with PBS (group D) for 1, 2, and 3 days; the expression levels of tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), and IL-6 were detected by ELISA. Results The results of Western blot showed that the expression of NFATc1 increased gradually with time, showing significant difference between different time points (P<0.05). However, after SC75741 was added, the expression of NFATc1 was inhibited at 2 and 3 days, showing significant difference when compared with no addition of SC75741 (P<0.001). After stimulation of PGN-sa, the expression of IkB-α decreased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes. Meanwhile, the expression of p-NF-κB increased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes; and the expression of p-NF-κB at 5 minutes was significantly higher than that at 10 minutes (P<0.001). After the addition of SC75741, there was no change in the expressions of IκB-α and p-NF-κB, showing no significant difference between different time points P>0.05). Moreover, the expressions of ERK, p38, JNK, NF-κB, Akt, p-p38, p-ERK, p-JNK, and p-Akt showed no significant change between different time points P>0.05). ELISA results showed that there were no expressions of TNF-α and IL-1α in groups A-D at different time points. The expression of IL-6 had an increasing trend with time prolonged in each group, showing significant differences between different time points (P<0.05). Moreover, at 1 day after culture, the expression of IL-6 showed no significant difference among groups P>0.05). At 2 and 3 days after culture, the expression of IL-6 in groups A-C showed an increasing trend and was significantly higher than that in group D, showing significant difference among groups (P<0.05). Conclusion PGN-sa can promote osteoclast differentiation through NF-κB signaling pathway, and IL-6 may play a role in this process.

Citation： REN Lirong, XU Yongqing, WANG Hai, HE Xiaoqing, SONG Muguo, CHEN Xueqiu. STUDY ON MOLECULAR MECHANISM OF OSTEOCLAST DIFFERENTIATION INDUCED BY STAPHYLOCOCCAL PEPTIDOGLYCAN. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(10): 1244-1248. doi: 10.7507/1002-1892.20160254 Copy

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8.	Tevlin R, McArdle A, Chan CK, et al. Osteoclast derivation from mouse bone marrow. J Vis Exp, 2014, (93):e52056.
9.	Martin TJ, Ng KW. Mechanisms by which cells of the osteoblast lineage control osteoclast formation and activity. J Cell Biochem, 1994, 56(3):357-366.
10.	Dougall WC, Glaccum M, Charrier K, et al. RANK is essential for osteoclast and lymph node development. Genes Dev, 1999, 13(18):2412-2424.
11.	Honma M, Ikebuchi Y, Kariya Y, et al. Regulatory mechanisms of RANKL presentation to osteoclast precursors. Curr Osteoporos Rep, 2014, 12(1):115-120.
12.	Soysa NS, Alles N, Aoki K, et al. Osteoclast formation and differentiation:an overview. J Med Dent Sci, 2012, 59(3):65-74.
13.	任莉荣, 徐永清. 破骨细胞分化机制的研究进展. 中国骨质疏松杂志, 2015, 21(12):1522-1525, 1528.
14.	Zhao Q, Wang X, Liu Y, et al. NFATc1:functions in osteoclasts. Int J Biochem Cell Biol, 2010, 42(5):576-579.
15.	Jimi E, Fukushima H. NF-kappaB signaling pathways and the future perspectives of bone disease therapy using selective inhibitors of NF-kappaB. Clin Calcium, 2016, 26(2):298-304.
16.	Schwandner R, Dziarski R, Wesche H, et al. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem, 1999, 274(25):17406-17409.
17.	Varoga D, Wruck CJ, Tohidnezhad M, et al. Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3. Histochem Cell Biol, 2009, 131(2):207-218.
18.	Algate K, Haynes DR, Bartold PM, et al. The effects of tumour necrosis factor-alpha on bone cells involved in periodontal alveolar bone loss; osteoclasts, osteoblasts and osteocytes. J Periodontal Res, 2016, 51(5):549-566.
19.	Boyce BF. Advances in the regulation of osteoclasts and osteoclast functions. J Dent Res, 2013, 92(10):860-867.
20.	Kudo O, Sabokbar A, Pocock A, et al. Interleukin-6 and interleukin-11 support human osteoclast formation by a RANKL-independent mechanism. Bone, 2003, 32(1):1-7.
21.	DiDonato JA, Mercurio F, Karin M. NF-kappaB and the link between inflammation and cancer. Immunol Rev, 2012, 246(1):379-400.

1. Montanaro L, Testoni F, Poggi A, et al. Emerging pathogenetic mechanisms of the implant-related osteomyelitis by Staphylococcus aureus. Int J Artif Organs, 2011, 34(9):781-788.
2. 黄金亮, 唐辉, 徐永清. 骨髓炎流行病学. 国际骨科学杂志, 2011, 32(2):94-95.
3. Fiedler T, Salamon A, Adam S, et al. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells. Exp Cell Res, 2013, 319(18):2883-2892.
4. Jin T, Zhu YL, Li J, et al. Staphylococcal protein A, Panton-Valentine leukocidin and coagulase aggravate the bone loss and bone destruction in osteomyelitis. Cell Physiol Biochem, 2013, 32(2):322-333.
5. Josse J, Velard F, Gangloff SC. Staphylococcus aureus vs. osteoblast:relationship and consequences in osteomyelitis. Front Cell Infect Microbiol, 2015, 5:85.
6. Trouillet-Assant S, Gallet M, Nauroy P, et al. Dual impact of live Staphylococcus aureus on the osteoclast lineage, leading to increased bone resorption. J Infect Dis, 2014, 211(4):571-581.
7. 任莉荣, 徐永清, 王海, 等. 金黄色葡萄球菌肽聚糖对破骨细胞分化的影响研究. 中国修复重建外科杂志, 2016, 30(8):1006-1010.
8. Tevlin R, McArdle A, Chan CK, et al. Osteoclast derivation from mouse bone marrow. J Vis Exp, 2014, (93):e52056.
9. Martin TJ, Ng KW. Mechanisms by which cells of the osteoblast lineage control osteoclast formation and activity. J Cell Biochem, 1994, 56(3):357-366.
10. Dougall WC, Glaccum M, Charrier K, et al. RANK is essential for osteoclast and lymph node development. Genes Dev, 1999, 13(18):2412-2424.
11. Honma M, Ikebuchi Y, Kariya Y, et al. Regulatory mechanisms of RANKL presentation to osteoclast precursors. Curr Osteoporos Rep, 2014, 12(1):115-120.
12. Soysa NS, Alles N, Aoki K, et al. Osteoclast formation and differentiation:an overview. J Med Dent Sci, 2012, 59(3):65-74.
13. 任莉荣, 徐永清. 破骨细胞分化机制的研究进展. 中国骨质疏松杂志, 2015, 21(12):1522-1525, 1528.
14. Zhao Q, Wang X, Liu Y, et al. NFATc1:functions in osteoclasts. Int J Biochem Cell Biol, 2010, 42(5):576-579.
15. Jimi E, Fukushima H. NF-kappaB signaling pathways and the future perspectives of bone disease therapy using selective inhibitors of NF-kappaB. Clin Calcium, 2016, 26(2):298-304.
16. Schwandner R, Dziarski R, Wesche H, et al. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem, 1999, 274(25):17406-17409.
17. Varoga D, Wruck CJ, Tohidnezhad M, et al. Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3. Histochem Cell Biol, 2009, 131(2):207-218.
18. Algate K, Haynes DR, Bartold PM, et al. The effects of tumour necrosis factor-alpha on bone cells involved in periodontal alveolar bone loss; osteoclasts, osteoblasts and osteocytes. J Periodontal Res, 2016, 51(5):549-566.
19. Boyce BF. Advances in the regulation of osteoclasts and osteoclast functions. J Dent Res, 2013, 92(10):860-867.
20. Kudo O, Sabokbar A, Pocock A, et al. Interleukin-6 and interleukin-11 support human osteoclast formation by a RANKL-independent mechanism. Bone, 2003, 32(1):1-7.
21. DiDonato JA, Mercurio F, Karin M. NF-kappaB and the link between inflammation and cancer. Immunol Rev, 2012, 246(1):379-400.

Chinese Journal of Reparative and Reconstructive Surgery

STUDY ON MOLECULAR MECHANISM OF OSTEOCLAST DIFFERENTIATION INDUCED BY STAPHYLOCOCCAL PEPTIDOGLYCAN

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