Rho信号通路在周期性张应力诱导人牙周膜细胞骨架重排中的作用
|
摘要
目的探讨周期性张应力对人牙周膜细胞(human periodontal ligament cells,hPDLCs)骨架重排的影响及Rho信号通路在其中的作用。方法应用FX-5000T细胞应变加载系统,对体外培养的hPDLCs施加周期性张应力,幅度为10%,频率为0.1 Hz,加载时间为6 h和24 h。以未加载的细胞作为对照组。应用倒置显微镜观察细胞形态变化,细胞免疫荧光染色和共聚焦显微镜观察细胞骨架蛋白F-actin的变化,并应用Western blot检测Rho通路相关蛋白的表达变化。用Rock的特异性抑制剂Y-27632预处理细胞后,在0.1 Hz,10%幅度条件下,加载6 h和24 h,检测细胞骨架蛋白F-actin的变化和Rho信号通路相关蛋白的表达变化。结果与静态组相比,周期性张应力诱导hPDLCs细胞骨架重排差异有统计学意义,Rho信号通路Rock和p-cofilin蛋白表达增加(P<0.05)。Rock的特异性抑制剂Y-27632可以降低Rock(P<0.05)和p-cofilin蛋白表达和细胞骨架重排。结论周期性张应力可促进体外培养的hPDLCs细胞骨架重排,Rho信号通路参与了此过程。
Objective To investigate the impact of cyclic strain on cytoskeletal arrangement of human periodontal ligament cells( hPDLCs) and the mechanism of Rho signaling pathway plays in it. Methods Ten percent-elongation of cyclic strain at 0. 1 Hz-frequency was applied to hPDLCs for 6 h or 24 h with FX-5000T system. The hPDLCs without cyclic strain under the same conditions were used as the control. The cell morphological changes were examined by phase-contrast microscopy. F-actin reorganization was observed by immunochemistry and confocal microscopy. Protein expression of Rho signaling pathway was analyzed by Western blot analysis. HPDLCs were preincubated with Y-27632,a specific Rock inhibitor,before cyclic strain application. F-actin reorganization was observed by immunochemistry and confocal microscopy,protein expression of Rho signaling pathway was also analyzed by Western blot analysis after 10%-elongation of cyclic strain at 0. 1 Hz-frequency was applied to hPDLCs for 6 h or 24 h. Results Cytoskeletal reorganization was observed after cyclic strain,compared to the static control. The strain also significantly increased Rock and p-cofilin expressions in hPDLCs( P < 0. 05). Additionally,Y-27632 devreased Rock and p-cofilin protein expressions( P < 0. 05) and cytoskeletal reorganization under cyclic strain. Conclusion Cyclic strain promotes cytoskeletal rearrangement of hPDLCs by Rho signaling pathway.
Objective To investigate the impact of cyclic strain on cytoskeletal arrangement of human periodontal ligament cells( hPDLCs) and the mechanism of Rho signaling pathway plays in it. Methods Ten percent-elongation of cyclic strain at 0. 1 Hz-frequency was applied to hPDLCs for 6 h or 24 h with FX-5000T system. The hPDLCs without cyclic strain under the same conditions were used as the control. The cell morphological changes were examined by phase-contrast microscopy. F-actin reorganization was observed by immunochemistry and confocal microscopy. Protein expression of Rho signaling pathway was analyzed by Western blot analysis. HPDLCs were preincubated with Y-27632,a specific Rock inhibitor,before cyclic strain application. F-actin reorganization was observed by immunochemistry and confocal microscopy,protein expression of Rho signaling pathway was also analyzed by Western blot analysis after 10%-elongation of cyclic strain at 0. 1 Hz-frequency was applied to hPDLCs for 6 h or 24 h. Results Cytoskeletal reorganization was observed after cyclic strain,compared to the static control. The strain also significantly increased Rock and p-cofilin expressions in hPDLCs( P < 0. 05). Additionally,Y-27632 devreased Rock and p-cofilin protein expressions( P < 0. 05) and cytoskeletal reorganization under cyclic strain. Conclusion Cyclic strain promotes cytoskeletal rearrangement of hPDLCs by Rho signaling pathway.
引文
[1]Wang JH,Thampatty BP.An introductory review of cell mechanobiology[J].Biomech Model Mechanobiol,2006,5(1):1-16.
[2]Sato R,Yamamoto H,Yamauchi M.Distribution pattern of versican,link protein and hyaluronic acid in the rat periodontal ligament during experimental tooth movement[J].J Periodontal Res,2002,37(1):15-22.
[3]Narayanan AS,Page RC.Connective tissues of the periodontium:a summary of current work[J].Coll Relat Res,1983,3(1):33-64.
[4]Mayr M,Hu Y,Hainaut H,et al.Mechanical stress-induced DNA damage and rac-p38MAPK signal pathways mediate p53-dependent apoptosis in vascular smooth muscle cells[J].FASEB J,2002,16(11):1423-1425.
[5]Li C,Xu Q.Mechanical stress-initiated signal transduction in vascular smooth muscle cells in vitro and in vivo[J].Cell Signal,2007,19(5):881-891.
[6]Qi YX,Qu MJ,Long DK,et al.Rho-GDP dissociation inhibitor alpha downregulated by low shear stress promotes vascular smooth muscle cell migration and apoptosis:a proteomic analysis[J].Cardiovasc Res,2008,80(1):114-122.
[7]Hall A.Rho GTPase and the actin cytoskeleton[J].Science,1998,279(5350):509-514.
[8]Bobik A.The structural basis of hypertension:vascular remodelling,rarefaction and angiogenesis/arteriogenesis[J].J Hypertens,2005,23(8):1473-1475.
[9]Archambault J,Tsuzaki M,Herzog W,et al.Stretch and interleukin-1 beta induce matrix metalloproteinases in rabbit tendon cells in vitro[J].J Orthop,Res,2002,20(1):36-39.
[10]Zeichen J,van Griensven M,Bosch U.The proliferative response of isolated human tendon fibroblasts to cyclic biaxial mechanical strain[J].Am J Sports Med,2000,28(6):888-892.
[11]Henneman S,Von den Hof JW,Maltha JC.Mechanobiology of tooth movement[J].Eur J Orthod,2008,30(3):299-306.
[12]Ingber DE.Mechanobiology and diseases of mechanotransduction[J].Ann Med,2003,35(8):564-577.
[13]Kawarizadeh A,Bourauel C,Gtz W,et al.Early responses of periodontal ligament cells to mechanical stimulus in vivo[J].J Dent Res,2005,84(10):902-906.
[14]Yano Y,Geibel J,Sumpio BE.Cyclic strain induces reorganization of integrin alpha 5 beta 1 and alpha 2 beta 1 in human umbilical vein endothelial cells[J].J Cell Biochem,1997,64(3):505-513.
[15]Yue H,Jinsong P,Xiaoli W,et al.Cyclic strain promotes migration and proliferation of human periodontal ligament cell via PI3K signaling pathway[J].Cell Mol Bioeng,2010,3(4):369-375.
[16]韩悦,潘劲松,陈丹鹏,等.ERK信号通路参与调控周期性张应力诱导的人牙周膜细胞增殖[J].医用生物力学,2009,24(3):211-222.
[17]Danciu TE,Gagari E,Adam RM,et al.Mechanical strain delivers anti-apoptotic and proliferative signals to gingival fibroblasts[J].J Dent Res,2004,83(8):596-601.
[18]Krishnan V,Davidovitch Z.On a path to unfolding the biological mechanisms of orthodontic tooth movement[J].J Dent Res,2009,88(7):597-608.
[19]Provenzano PP,Keely PJ.Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling[J].J Cell Science,2012,124(8):1195-1205.
[20]Morioka M,Parameswaran H,Naruse K,et al.Microtubule dynamics regulate cyclic stretch-induced cell alignment in human airway smooth muscle cells[J].PLoS ONE,2011,6(10):26384.
[21]Revenu C,Athman R,Robine S,et al.The co-workers of actin filaments:from cell structures to signals[J].Nat Rev Mol Cell Biol,2004,5(8):635-6426.
[22]Woodring PJ,Hunter T,Wang JY.Regulation of F-actin-dependent processes by the Abl family of tyrosine kinases[J].J Cell Sci,2003,116(13):2613-2626.
[23]van Nieuw Amerongen GP,van Hinsbergh VW.Cytoskeletal effects of rho-like small guanine nucleotide-binding proteins in the vascular system[J].Arterioscler Thromb Vasc Biol,2001,21(3):300-311.
[24]Ohtsu H,Mifune M,Frank GD,et al.Signal-crosstalk between Rho/Rock and c-Jun NH2-term-inal kinase mediates migration of vascular smooth muscle cells stimulated by angiotensin II[J].Arterioscler Thromb Vasc Biol,2005,25(9):1831-1836.
[25]Onoue N,Nawata J,Tada T,et al.Increased static pressure promotes migration of vascular smooth muscle cells:involvement of the Rho-kinase pathway[J].J Cardiovasc Pharmacol,2008,51(1):55-61.
[2]Sato R,Yamamoto H,Yamauchi M.Distribution pattern of versican,link protein and hyaluronic acid in the rat periodontal ligament during experimental tooth movement[J].J Periodontal Res,2002,37(1):15-22.
[3]Narayanan AS,Page RC.Connective tissues of the periodontium:a summary of current work[J].Coll Relat Res,1983,3(1):33-64.
[4]Mayr M,Hu Y,Hainaut H,et al.Mechanical stress-induced DNA damage and rac-p38MAPK signal pathways mediate p53-dependent apoptosis in vascular smooth muscle cells[J].FASEB J,2002,16(11):1423-1425.
[5]Li C,Xu Q.Mechanical stress-initiated signal transduction in vascular smooth muscle cells in vitro and in vivo[J].Cell Signal,2007,19(5):881-891.
[6]Qi YX,Qu MJ,Long DK,et al.Rho-GDP dissociation inhibitor alpha downregulated by low shear stress promotes vascular smooth muscle cell migration and apoptosis:a proteomic analysis[J].Cardiovasc Res,2008,80(1):114-122.
[7]Hall A.Rho GTPase and the actin cytoskeleton[J].Science,1998,279(5350):509-514.
[8]Bobik A.The structural basis of hypertension:vascular remodelling,rarefaction and angiogenesis/arteriogenesis[J].J Hypertens,2005,23(8):1473-1475.
[9]Archambault J,Tsuzaki M,Herzog W,et al.Stretch and interleukin-1 beta induce matrix metalloproteinases in rabbit tendon cells in vitro[J].J Orthop,Res,2002,20(1):36-39.
[10]Zeichen J,van Griensven M,Bosch U.The proliferative response of isolated human tendon fibroblasts to cyclic biaxial mechanical strain[J].Am J Sports Med,2000,28(6):888-892.
[11]Henneman S,Von den Hof JW,Maltha JC.Mechanobiology of tooth movement[J].Eur J Orthod,2008,30(3):299-306.
[12]Ingber DE.Mechanobiology and diseases of mechanotransduction[J].Ann Med,2003,35(8):564-577.
[13]Kawarizadeh A,Bourauel C,Gtz W,et al.Early responses of periodontal ligament cells to mechanical stimulus in vivo[J].J Dent Res,2005,84(10):902-906.
[14]Yano Y,Geibel J,Sumpio BE.Cyclic strain induces reorganization of integrin alpha 5 beta 1 and alpha 2 beta 1 in human umbilical vein endothelial cells[J].J Cell Biochem,1997,64(3):505-513.
[15]Yue H,Jinsong P,Xiaoli W,et al.Cyclic strain promotes migration and proliferation of human periodontal ligament cell via PI3K signaling pathway[J].Cell Mol Bioeng,2010,3(4):369-375.
[16]韩悦,潘劲松,陈丹鹏,等.ERK信号通路参与调控周期性张应力诱导的人牙周膜细胞增殖[J].医用生物力学,2009,24(3):211-222.
[17]Danciu TE,Gagari E,Adam RM,et al.Mechanical strain delivers anti-apoptotic and proliferative signals to gingival fibroblasts[J].J Dent Res,2004,83(8):596-601.
[18]Krishnan V,Davidovitch Z.On a path to unfolding the biological mechanisms of orthodontic tooth movement[J].J Dent Res,2009,88(7):597-608.
[19]Provenzano PP,Keely PJ.Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling[J].J Cell Science,2012,124(8):1195-1205.
[20]Morioka M,Parameswaran H,Naruse K,et al.Microtubule dynamics regulate cyclic stretch-induced cell alignment in human airway smooth muscle cells[J].PLoS ONE,2011,6(10):26384.
[21]Revenu C,Athman R,Robine S,et al.The co-workers of actin filaments:from cell structures to signals[J].Nat Rev Mol Cell Biol,2004,5(8):635-6426.
[22]Woodring PJ,Hunter T,Wang JY.Regulation of F-actin-dependent processes by the Abl family of tyrosine kinases[J].J Cell Sci,2003,116(13):2613-2626.
[23]van Nieuw Amerongen GP,van Hinsbergh VW.Cytoskeletal effects of rho-like small guanine nucleotide-binding proteins in the vascular system[J].Arterioscler Thromb Vasc Biol,2001,21(3):300-311.
[24]Ohtsu H,Mifune M,Frank GD,et al.Signal-crosstalk between Rho/Rock and c-Jun NH2-term-inal kinase mediates migration of vascular smooth muscle cells stimulated by angiotensin II[J].Arterioscler Thromb Vasc Biol,2005,25(9):1831-1836.
[25]Onoue N,Nawata J,Tada T,et al.Increased static pressure promotes migration of vascular smooth muscle cells:involvement of the Rho-kinase pathway[J].J Cardiovasc Pharmacol,2008,51(1):55-61.