CFTR在雌激素诱导破骨细胞凋亡中的作用机制研究
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摘要
目的探讨囊性纤维化跨膜转导调节因子(CFTR)在雌激素诱导破骨细胞凋亡中的可能作用机制。方法用不同浓度雌激素作用体外培养的人破骨细胞,采用Western Blot法检测不同时间点(6、12、24 h)CFTR蛋白水平表达变化,获得最合适浓度及最佳作用时间。在最合适浓度雌激素刺激下加入CFTR抑制剂下调CFTR的表达,用流式技术检测其对破骨细胞凋亡的影响。通过雌激素、雌激素受体阻滞剂处理破骨细胞,采用Western Blot法检测雌激素受体ERα、ERβ、CFTR及凋亡相关蛋白Fas/FasL的表达水平。结果当1×10~(-7) mol/L雌激素作用24 h时CFTR的表达水平较其他浓度及其他时间点高,加入CFTR抑制剂后可显著下调CFTR的表达水平。加入雌激素后,破骨细胞的凋亡率显著升高,加入抑制剂后,可以逆转凋亡率升高。加入雌激素后,雌激素受体ERα和ERβ的表达水平升高,而且促进CFTR的表达;加入ER阻滞剂后,逆转了雌激素对其受体及CFTR的高表达。加入雌激素诱导后,FasL及Fas蛋白的表达水平升高,加入ER阻滞剂可以逆转雌激素诱导的凋亡相关蛋白表达水平升高。结论雌激素通过雌激素受体上调CFTR的表达进而诱导破骨细胞凋亡。
Objective To study the role and mechanism of cystic fibrosis transmembrane conductance regulator(CFTR) in estrogen-induced apoptosis of osteoclasts. Methods Human osteoclasts treated with different concentrations of estrogen were cultured in vitro at indicated times. Western blot assay was performed to detect the CFTR protein level as to choose the optimal concentration and the best effect time(6, 12, 24 h). Under the optimal concentration of estrogen stimulation, CFTR inhibitor was added to down-regulate the expression of CFTR, and flow cytometry was used to evaluate its effect on osteoclast apoptosis. Moreover, osteoclasts were treated with estrogen and estrogen receptor blockers, and western blot was adapted to detect the expression levels of estrogen receptor ERα, ERβ, CFTR, and apoptosis-related proteins Fas/FasL. Results The expression of CFTR was significantly increased after osteoclasts were treated with estrogen 1 ×10~(-7) mol/L for 24 h, while CFTR inhibitor could notably decease the expression level of CFTR. In addition, estrogen had the positive activity on promoting the apoptosis of osteoclasts, and on the contrary, the effects of estrogen on inducing apoptosis of osteoclasts were remarkably reversed after treatment with CFTR inhibitor. Besides, after treatment with estrogen, the expression levels of estrogen receptors ERα and ERβ were obviously up-regulated, and the expression of CFTR was also up-regulated. However, the addition of estrogen receptor blocker reversed the high expression levels of estrogen to its receptors and CFTR. Moreover, after induction of estrogen, the expression levels of FasL and Fas were increased, and the addition of estrogen receptor could reverse the increase of apoptosis-related proteins induced by estrogen. Conclusion Estrogen induces apoptosis of osteoclasts via up-regulation of CFTR through estrogen receptors.
Objective To study the role and mechanism of cystic fibrosis transmembrane conductance regulator(CFTR) in estrogen-induced apoptosis of osteoclasts. Methods Human osteoclasts treated with different concentrations of estrogen were cultured in vitro at indicated times. Western blot assay was performed to detect the CFTR protein level as to choose the optimal concentration and the best effect time(6, 12, 24 h). Under the optimal concentration of estrogen stimulation, CFTR inhibitor was added to down-regulate the expression of CFTR, and flow cytometry was used to evaluate its effect on osteoclast apoptosis. Moreover, osteoclasts were treated with estrogen and estrogen receptor blockers, and western blot was adapted to detect the expression levels of estrogen receptor ERα, ERβ, CFTR, and apoptosis-related proteins Fas/FasL. Results The expression of CFTR was significantly increased after osteoclasts were treated with estrogen 1 ×10~(-7) mol/L for 24 h, while CFTR inhibitor could notably decease the expression level of CFTR. In addition, estrogen had the positive activity on promoting the apoptosis of osteoclasts, and on the contrary, the effects of estrogen on inducing apoptosis of osteoclasts were remarkably reversed after treatment with CFTR inhibitor. Besides, after treatment with estrogen, the expression levels of estrogen receptors ERα and ERβ were obviously up-regulated, and the expression of CFTR was also up-regulated. However, the addition of estrogen receptor blocker reversed the high expression levels of estrogen to its receptors and CFTR. Moreover, after induction of estrogen, the expression levels of FasL and Fas were increased, and the addition of estrogen receptor could reverse the increase of apoptosis-related proteins induced by estrogen. Conclusion Estrogen induces apoptosis of osteoclasts via up-regulation of CFTR through estrogen receptors.
引文
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[3]Laube M,Kuppers E,Thome UH.Modulation of sodium transport in alveolar epithelial cells by estradiol and progesterone[J].Pediatr Res,2011,69(3):200-205.
[4]Bien K,Sokolowska J,Baska P,et al.Fas/FasL pathway participates in regulation of antiviral and inflammatory response during mousepox infection of lungs[J].Mediators Inflamm,2015,2015:281613.
[5]Nakamura T,Imai Y,Matsumoto T,et al.Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts[J].Cell,2007,130(5):811-823.
[6]Frenkel B,Hong A,Baniwal SK,et al.Regulation of adult bone turnover by sex steroids[J].J Cell Physiol,2010,224(2):305-310.
[7]Kovacic N,Lukic IK,Grcevic D,et al.The Fas/Fas ligand system inhibits differentiation of murine osteoblasts but has a limited role in osteoblast and osteoclast apoptosis[J].J Immunol,2007,178(6):3379-3389.
[8]Marusic A,Kovacic N,Lukic IK,et al.Understanding the role of Fas-Fas ligand system in bone[J].Arthritis Res Ther,2012,14(Suppl 1):20.
[9]Katavic V,Lukic IK,Kovacic N,et al.Increased bone mass is a part of the generalized lymphoproliferative disorder phenotype in the mouse[J].J Immunol,2003,170(3):1540-1547.
[10]Wang L,Liu S,Zhao Y,et al.Osteoblast-induced osteoclast apoptosis by fas ligand/FAS pathway is required for maintenance of bone mass[J].Cell Death Differ,2015,22(10):1654-1664.
[11]Nakamura T,Imai Y,Matsumoto T,et al.Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts[J].Cell,2007,130(5):811-823.