Effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHENG Libin ¹ ,  HUANG Dong ^1,2

1. Guangdong Medical University, Zhanjiang Guangdong, 524023, P.R.China;
2. Department of Trauma Orthopedics, Guangdong Second Provincial Central Hospital, Guangzhou Guangdong, 510000, P.R.China;

Corresponding author：

HUANG Dong, Email: dong_177@126.com

Keywords：

FTY720-P; EphA2; EphrinA2; RhoA; osteoclasts

DOI：

10.7507/1002-1892.201710109

Video：

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ObjectiveTo investigate the effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts.MethodsMurine RAW264.7 macrophages were induced into osteoclasts by dexamethasone and 1α, 25-dihydroxyvitamin D₃, and identified by tartrate resistant acid phosphatase (TRAP) staining. Then, the osteoclasts were divided into 2 groups. The osteoclasts were treated with 400 ng/mL FTY720-P in experimental group and without FTY720-P in control group, respectively. After 48 hours of culture, the cells in 2 groups were detected by real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining. The expressions of EphA2, EphrinA2, RhoA, and the bone reconstruction associated proteins［bone morphogenetic protein 2 (BMP-2) and transform growth factor β₁ (TGF-β₁)］were analyzed and compared.ResultsRAW264.7 cells were successfully induced into osteoclasts identified by TRAP staining. Compared with control group, the relative expressions of EphA2 and EphrinA2 mRNAs and proteins in experimental group significantly decreased after 48 hours (P<0.05), and the relative expression of RhoA protein also significantly decreased (P<0.05). The relative expressions of BMP-2 and TGF-β₁ mRNAs were significantly increased (P<0.05), and those protein expressions were enhanced.ConclusionFTY720-P can down-regulate the expression of RhoA and promote the expressions of TGF- β₁ and BMP-2 by affecting the transduction of EphA2-EphrinA2 bidirectional signaling in osteoclasts.

Citation： ZHENG Libin, HUANG Dong. Effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(5): 575-580. doi: 10.7507/1002-1892.201710109 Copy

1.	Irie N, Takada Y, Watanabe Y, et al. Bidirectional signaling through ephrinA2-EphA2 enhances osteoclastogenesis and suppresses osteoblastogenesis. J Biol Chem, 2009, 284(21): 14637-14644.
2.	Stark DA, Coffey NJ, Pancoast HR, et al. Ephrin-A3 promotes and maintains slow muscle fiber identity during postnatal development and reinnervation. J Cell Biol, 2015, 211(5): 1077-1091.
3.	Xu X, Gao Y, Shan F, et al. A novel role for RGMa in modulation of bone marrow-derived dendritic cells maturation induced by lipopolysaccharide. International Immunopharmacology, 2016, 33 (4): 99-107.
4.	Dines M, Grinberg S, Vassiliev M, et al. The roles of Eph receptors in contextual fear conditioning memory formation. Neurobiol Learn Mem, 2015, 124: 62-70.
5.	Martin TJ, Allan EH, Ho PW, et al. Communication between ephrinB2 and EphB4 within the osteoblast lineage. Adv Exp Med Biol, 2010, 658: 51-60.
6.	Xing L, Son JH, Stevenson TJ, et al. A serotonin circuit acts as an environmental sensor to mediate midline axon crossing through EphrinB2. J Neurosci, 2015, 35 (44): 14794-14808.
7.	Klein R. Eph/ephrin signaling in morphogenesis, neural development and plasticity. Curr Opin Cell Biol, 2004, 16(5): 580-589.
8.	Serrano MJ, Liu J, Svoboda KK, et al. Ephrin reverse signaling mediates palatal fusion and epithelial-to-mesenchymal transition independently of Tgfβ3. J Cell Physiol, 2015, 230 (12): 2961-2972.
9.	Nunan R, Campbell J, Mori R, et al. Ephrin-Bs drive junctional downregulation and actin stress fiber disassembly to enable wound re-epithelialization. Cell Rep, 2015, 13 (7): 1380-1395.
10.	Egea J, Klein R. Bidirectional Eph-ephrin signaling during axon guidance. Trends Cell Biol, 2007, 17(5): 230-238.
11.	Luessi F, Kraus S, Trinschek B, et al. FTY720 (fingolimod) treatment tips the balance towards less immunogenic antigen-presenting cells in patients with multiple sclerosis. Mult Scler, 2015, 21 (14): 1811-1822.
12.	De Roo AK, Struyf S, Foets B, et al. Transforming growth factor beta switch in aqueous humor of patients with Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci, 2016, 57 (2): 771-772.
13.	Das A, Barker DA, Wang T, et al. Delivery of bioactive lipids from composite microgel-microsphere injectable scaffolds enhances stem cell recruitment and skeletal repair. PLoS One, 2014, 9(7): e101276.
14.	Zhang D, Huang Y, Huang Z, et al. FTY-720P suppresses osteoclast formation by regulating expression of interleukin-6 (IL-6), interleukin-4 (IL-4), and matrix metalloproteinase 2 (MMP-2). Med Sci Monit, 2016, 22: 2187-2194.
15.	陈智颖, 左洁仪, 兰万利, 等. FTY720-P 对成骨细胞分化成熟的作用. 广东医学, 2017, 38(13): 1957-1961.
16.	Mundy GR, Elefteriou F. Boning up on ephrin signaling. Cell, 2006, 126(3): 441-443.
17.	Takeuchi S, Katoh H, Negishi M. Eph/ephrin reverse signalling induces axonal retraction through RhoA/ROCK pathway. J Biochem, 2015, 158(3): 245-252.
18.	Diercke K, Sen S, Kohl A, et al. Compression-dependent up-regulation of ephrin-A2 in PDL fibroblasts attenuates osteogenesis. J Dent Res, 2011, 90(9): 1108-1115.
19.	熊苗, 朱洁萍, 李莉, 等. 过继转移 FTY720-DC 对自然流产模型孕鼠胚胎丢失率的影响. 上海交通大学学报 (医学版), 2016, 36(6): 793-798.
20.	De Roo AK, Struyf S, Foets B, et al. Transforming growth factor beta switch in aqueous humor of patients with Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci, 2016, 57 (2): 771-772.
21.	Matthaei M, Gillessen J, Muether PS, et al. Author response: transforming growth factor beta switch in aqueous humor of patients with Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci, 2016, 57 (2): 773.
22.	Brili S, Antonopoulos AS, Oikonomou E, et al. Impairment of arterial elastic properties and elevated circulating levels of transforming growth factor-beta in subjects with repaired coarctation of aorta. Int J Cardiol, 2016, 207: 282-283.
23.	Mirakbari SM. Proposal for a new mechanism of action for aluminum phosphide (ALP) for causing local injuries in ALP poisoning: Should treatment strategies be modified? Hum Expe Toxicol, 2016, 35 (10): 1145-1146.
24.	Schumacher TC, Aminian A, Volkmann E, et al. Synthesis and mechanical evaluation of Sr-doped calcium-zirconium-silicate (baghdadite) and its impact on osteoblast cell proliferation and ALP activity. Biomedical Materials (Bristol, England), 2015, 10 (5): 055013.

1. Irie N, Takada Y, Watanabe Y, et al. Bidirectional signaling through ephrinA2-EphA2 enhances osteoclastogenesis and suppresses osteoblastogenesis. J Biol Chem, 2009, 284(21): 14637-14644.
2. Stark DA, Coffey NJ, Pancoast HR, et al. Ephrin-A3 promotes and maintains slow muscle fiber identity during postnatal development and reinnervation. J Cell Biol, 2015, 211(5): 1077-1091.
3. Xu X, Gao Y, Shan F, et al. A novel role for RGMa in modulation of bone marrow-derived dendritic cells maturation induced by lipopolysaccharide. International Immunopharmacology, 2016, 33 (4): 99-107.
4. Dines M, Grinberg S, Vassiliev M, et al. The roles of Eph receptors in contextual fear conditioning memory formation. Neurobiol Learn Mem, 2015, 124: 62-70.
5. Martin TJ, Allan EH, Ho PW, et al. Communication between ephrinB2 and EphB4 within the osteoblast lineage. Adv Exp Med Biol, 2010, 658: 51-60.
6. Xing L, Son JH, Stevenson TJ, et al. A serotonin circuit acts as an environmental sensor to mediate midline axon crossing through EphrinB2. J Neurosci, 2015, 35 (44): 14794-14808.
7. Klein R. Eph/ephrin signaling in morphogenesis, neural development and plasticity. Curr Opin Cell Biol, 2004, 16(5): 580-589.
8. Serrano MJ, Liu J, Svoboda KK, et al. Ephrin reverse signaling mediates palatal fusion and epithelial-to-mesenchymal transition independently of Tgfβ3. J Cell Physiol, 2015, 230 (12): 2961-2972.
9. Nunan R, Campbell J, Mori R, et al. Ephrin-Bs drive junctional downregulation and actin stress fiber disassembly to enable wound re-epithelialization. Cell Rep, 2015, 13 (7): 1380-1395.
10. Egea J, Klein R. Bidirectional Eph-ephrin signaling during axon guidance. Trends Cell Biol, 2007, 17(5): 230-238.
11. Luessi F, Kraus S, Trinschek B, et al. FTY720 (fingolimod) treatment tips the balance towards less immunogenic antigen-presenting cells in patients with multiple sclerosis. Mult Scler, 2015, 21 (14): 1811-1822.
12. De Roo AK, Struyf S, Foets B, et al. Transforming growth factor beta switch in aqueous humor of patients with Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci, 2016, 57 (2): 771-772.
13. Das A, Barker DA, Wang T, et al. Delivery of bioactive lipids from composite microgel-microsphere injectable scaffolds enhances stem cell recruitment and skeletal repair. PLoS One, 2014, 9(7): e101276.
14. Zhang D, Huang Y, Huang Z, et al. FTY-720P suppresses osteoclast formation by regulating expression of interleukin-6 (IL-6), interleukin-4 (IL-4), and matrix metalloproteinase 2 (MMP-2). Med Sci Monit, 2016, 22: 2187-2194.
15. 陈智颖, 左洁仪, 兰万利, 等. FTY720-P 对成骨细胞分化成熟的作用. 广东医学, 2017, 38(13): 1957-1961.
16. Mundy GR, Elefteriou F. Boning up on ephrin signaling. Cell, 2006, 126(3): 441-443.
17. Takeuchi S, Katoh H, Negishi M. Eph/ephrin reverse signalling induces axonal retraction through RhoA/ROCK pathway. J Biochem, 2015, 158(3): 245-252.
18. Diercke K, Sen S, Kohl A, et al. Compression-dependent up-regulation of ephrin-A2 in PDL fibroblasts attenuates osteogenesis. J Dent Res, 2011, 90(9): 1108-1115.
19. 熊苗, 朱洁萍, 李莉, 等. 过继转移 FTY720-DC 对自然流产模型孕鼠胚胎丢失率的影响. 上海交通大学学报 (医学版), 2016, 36(6): 793-798.
20. De Roo AK, Struyf S, Foets B, et al. Transforming growth factor beta switch in aqueous humor of patients with Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci, 2016, 57 (2): 771-772.
21. Matthaei M, Gillessen J, Muether PS, et al. Author response: transforming growth factor beta switch in aqueous humor of patients with Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci, 2016, 57 (2): 773.
22. Brili S, Antonopoulos AS, Oikonomou E, et al. Impairment of arterial elastic properties and elevated circulating levels of transforming growth factor-beta in subjects with repaired coarctation of aorta. Int J Cardiol, 2016, 207: 282-283.
23. Mirakbari SM. Proposal for a new mechanism of action for aluminum phosphide (ALP) for causing local injuries in ALP poisoning: Should treatment strategies be modified? Hum Expe Toxicol, 2016, 35 (10): 1145-1146.
24. Schumacher TC, Aminian A, Volkmann E, et al. Synthesis and mechanical evaluation of Sr-doped calcium-zirconium-silicate (baghdadite) and its impact on osteoblast cell proliferation and ALP activity. Biomedical Materials (Bristol, England), 2015, 10 (5): 055013.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts

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