ROCK亚型在血管平滑肌细胞迁移和增殖中的作用
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摘要
目的:血管平滑肌细胞的迁移和增殖在动脉粥样硬化、血管术后再狭窄等心血管疾病中具有重要作用。影响血管平滑肌细胞的迁移和增殖的信号转导通路很多,其中在RhoA/ROCK信号转导通路中,ROCK通过使其下游的蛋白质磷酸化,间接地调控肌动蛋白的聚合和解聚以及与肌球蛋白的相互作用,最终影响细胞的迁移。研究还发现ROCK能通过调节细胞周期蛋白的表达来调控细胞周期的进行,从而影响血管平滑肌细胞的增殖。ROCK有两种亚型包括ROCK1和ROCK2,两者的氨基酸序列具有65%的同源性,其中激酶区的同源性高达92%。因此,一般认为两者在细胞内的功能相同。然而,近年来研究发现ROCK1和ROCK2在某些细胞中具有不同的磷酸化底物和细胞功能。目前,ROCK的两种亚型在血管平滑肌细胞的迁移和增殖的作用是否相同尚不清楚。本研究旨在研究ROCK亚型(ROCK1和ROCK2)在血管平滑肌细胞迁移和增殖中的作用及其分子机制。
方法:(1)利用RNA干扰原理,通过脂质体转染特异性针对ROCK亚型的人工合成的siRNA,使ROCK1和ROCK2表达分别下调,观察ROCK1和ROCK2对PDGF诱导的血管平滑肌细胞(A7r5)迁移和血清刺激的增殖是否具有相同的作用;(2)使用ROCK的特异抑制剂Y-27632同时抑制ROCK1和ROCK2的激酶活性,观察ROCK对细胞迁移和增殖的影响;(3)用蛋白免疫印迹技术检测蛋白的表达情况;(4)使用Boyden Chamber法观察ROCK1和ROCK2表达下调后对血管平滑肌细胞迁移的影响;(5)使用MTT法检测ROCK激酶表达下调后血管平滑肌细胞的生长曲线,并用流式细胞仪通过PI染色法分析经血清饥饿法同步化的细胞中各细胞周期时相的变化。
结果:(1)ROCK1和ROCK2在血管平滑肌细胞中的蛋白表达水平不同,ROCK2的表达水平是ROCK1的8倍;(2)做细胞迁移实验时,通过转染siRNA使ROCK1和ROCK2的表达分别下调83.4%和94.7%;(3)ROCK1表达下调后,抑制了血管平滑肌细胞的迁移,而ROCK2表达下调后对细胞的迁移无明显影响,ROCK活性被Y-27632抑制后也抑制了细胞的迁移;(4)ROCK1表达下调导致LIMK的磷酸化水平降低,ROCK2表达下调后对LIMK磷酸化水平无明显影响,Y-27632抑制ROCK活性后LIMK的磷酸化水平也降低;(5)ROCK1表达下调后导致MLC磷酸化水平增加,ROCK2表达下调后对MLC磷酸化水平无明显影响,Y-27632抑制ROCK活性也可使MLC磷酸化水平也增加;(6)转染siRNA后,ROCK1和ROCK2两组之间细胞的生长无明显差别,而且细胞周期G1/S期的转化也无明显变化,而Y-27632抑制ROCK活性后G1/S期的转化受到抑制。
结论:(1)在ROCK的两种亚型中,ROCK1在血管平滑肌细胞的迁移中起了主导作用,ROCK2对血管平滑肌细胞的迁移的调节作用不明显;(2)ROCK1通过下调LIMK的磷酸化,上调MLC的磷酸化途径调控血管平滑肌细胞的迁移;(3)ROCK1和ROCK2对血管平滑肌细胞的增殖具有相同的作用。
Objectives:The migration and proliferation of vascular smooth muscle cells(VSMCs) play important roles in cardiovascular diseases such as ather- osclerosis, vascular restenosis. There are many signaling pathways in which impact the VSMCs migration and proliferation. In the RhoA/ROCK pathway, ROCK indirectly regulates actin polymerization and actomyosin contracti- lity through its downstream protein phosphorylation. Consequently, ROCK affects cell migration. The previous study found that ROCK can also control the progression of the cell cycle by regulating the expression of cell cycle proteins, thus affecting VSMCs proliferation. There are two ROCK informs including ROCK1 and ROCK2, that share overall 65% homology in amino acid level, in which the kinase domain homology approached as high as 92%. Therefore, it is generally believed that they have same functions in the cells. However, recent study found that ROCK1 and ROCK2 in some cells have different substrates or cell functions. At present, it is not clear that whether they have the same effects of the ROCK informs on VSMCs migration and proliferation. This study is aimed to examine the effects of ROCK1 and ROCK2 on VSMCs migration and proliferation, as well as their molecular mechanisms.
Methods:(1)Based on the RNA interference theory, transfection synthetic siRNA targeting specifically ROCK isoforms by liposome, so that the expression of ROCK1 or ROCK2 was down regulated respectively, in order for further observation the effects of ROCK1 and ROCK2 on VSMCs migration and proliferation.(2)Using the specific ROCK inhibitor Y-27632 that inhibits the ROCK1 and ROCK2 kinase activity, to observe the effects of ROCK on VSMCs migration and proliferation.(3)To detect the expression of various downstream proteins of ROCK, Western blotting was used.(4)To observe the effets of ROCK1 and ROCK2 on vascular smooth muscle cell migration, Boyden Chamber assay was used.(5)To detect the effects of ROCK1 and ROCK2 on vascular smooth muscle cell proliferation, MTT assay was used, and flow cytometry was adopted for analysis of cell cycle phase progression by PI staining method after serum starvation to synchroniz cells.
Results:(1)There were different expression levels between ROCK1 and ROCK2 in VSMCs, the expression of ROCK2 is higher eight times than that of ROCK1.(2)By using siRNA transfection, the expression of ROCK1 and ROCK2 were down regulated by 83.4% and 94.7% respectively as the cell migration experiments.(3)When reduced the expression of ROCK1, the migration of VSMCs was inhibited, but ROCK2 had no significant effect on VSMCs migration at the same condition. Also,ROCK activity blocked by Y-27632 also inhibited the VSMCs migration.(4)ROCK1 downregulation resulted in reduced levels of LIMK1 and LIMK2 phosphorylation, and ROCK2 had no effects on phosphorylation of LIMK1 and LIMK2. In addition, LIMK phosphorylation decreased after ROCK activity inhibition by Y-27632.(5)ROCK1 downregulation led to increased level of MLC phosphorylation, and ROCK2 had no significant effect on the level of MLC phosphorylation ROCK activity inhibition by Y-27632 also increased the level of MLC phosphorylation.(6)When ROCK1 and ROCK2 were down regulated respectively, no significant difference was observed in cell growth, and the cell cycle G1/S phase was also not altered obviously, whereas ROCK activity inhibition by Y-27632 leads to the inhibition of the G1/S phase progress.
Conclusions:(1)In the two ROCK informs, ROCK1, but not ROCK2, plays a leading role in VSMCs migration.(2)ROCK1 controls the migration of VSMCs via reducing the levels of LIMK phosphorylation and increasing MLC phosphorylation.(3)ROCK1 and ROCK2 have the same effects on the proliferation of VSMCs.
方法:(1)利用RNA干扰原理,通过脂质体转染特异性针对ROCK亚型的人工合成的siRNA,使ROCK1和ROCK2表达分别下调,观察ROCK1和ROCK2对PDGF诱导的血管平滑肌细胞(A7r5)迁移和血清刺激的增殖是否具有相同的作用;(2)使用ROCK的特异抑制剂Y-27632同时抑制ROCK1和ROCK2的激酶活性,观察ROCK对细胞迁移和增殖的影响;(3)用蛋白免疫印迹技术检测蛋白的表达情况;(4)使用Boyden Chamber法观察ROCK1和ROCK2表达下调后对血管平滑肌细胞迁移的影响;(5)使用MTT法检测ROCK激酶表达下调后血管平滑肌细胞的生长曲线,并用流式细胞仪通过PI染色法分析经血清饥饿法同步化的细胞中各细胞周期时相的变化。
结果:(1)ROCK1和ROCK2在血管平滑肌细胞中的蛋白表达水平不同,ROCK2的表达水平是ROCK1的8倍;(2)做细胞迁移实验时,通过转染siRNA使ROCK1和ROCK2的表达分别下调83.4%和94.7%;(3)ROCK1表达下调后,抑制了血管平滑肌细胞的迁移,而ROCK2表达下调后对细胞的迁移无明显影响,ROCK活性被Y-27632抑制后也抑制了细胞的迁移;(4)ROCK1表达下调导致LIMK的磷酸化水平降低,ROCK2表达下调后对LIMK磷酸化水平无明显影响,Y-27632抑制ROCK活性后LIMK的磷酸化水平也降低;(5)ROCK1表达下调后导致MLC磷酸化水平增加,ROCK2表达下调后对MLC磷酸化水平无明显影响,Y-27632抑制ROCK活性也可使MLC磷酸化水平也增加;(6)转染siRNA后,ROCK1和ROCK2两组之间细胞的生长无明显差别,而且细胞周期G1/S期的转化也无明显变化,而Y-27632抑制ROCK活性后G1/S期的转化受到抑制。
结论:(1)在ROCK的两种亚型中,ROCK1在血管平滑肌细胞的迁移中起了主导作用,ROCK2对血管平滑肌细胞的迁移的调节作用不明显;(2)ROCK1通过下调LIMK的磷酸化,上调MLC的磷酸化途径调控血管平滑肌细胞的迁移;(3)ROCK1和ROCK2对血管平滑肌细胞的增殖具有相同的作用。
Objectives:The migration and proliferation of vascular smooth muscle cells(VSMCs) play important roles in cardiovascular diseases such as ather- osclerosis, vascular restenosis. There are many signaling pathways in which impact the VSMCs migration and proliferation. In the RhoA/ROCK pathway, ROCK indirectly regulates actin polymerization and actomyosin contracti- lity through its downstream protein phosphorylation. Consequently, ROCK affects cell migration. The previous study found that ROCK can also control the progression of the cell cycle by regulating the expression of cell cycle proteins, thus affecting VSMCs proliferation. There are two ROCK informs including ROCK1 and ROCK2, that share overall 65% homology in amino acid level, in which the kinase domain homology approached as high as 92%. Therefore, it is generally believed that they have same functions in the cells. However, recent study found that ROCK1 and ROCK2 in some cells have different substrates or cell functions. At present, it is not clear that whether they have the same effects of the ROCK informs on VSMCs migration and proliferation. This study is aimed to examine the effects of ROCK1 and ROCK2 on VSMCs migration and proliferation, as well as their molecular mechanisms.
Methods:(1)Based on the RNA interference theory, transfection synthetic siRNA targeting specifically ROCK isoforms by liposome, so that the expression of ROCK1 or ROCK2 was down regulated respectively, in order for further observation the effects of ROCK1 and ROCK2 on VSMCs migration and proliferation.(2)Using the specific ROCK inhibitor Y-27632 that inhibits the ROCK1 and ROCK2 kinase activity, to observe the effects of ROCK on VSMCs migration and proliferation.(3)To detect the expression of various downstream proteins of ROCK, Western blotting was used.(4)To observe the effets of ROCK1 and ROCK2 on vascular smooth muscle cell migration, Boyden Chamber assay was used.(5)To detect the effects of ROCK1 and ROCK2 on vascular smooth muscle cell proliferation, MTT assay was used, and flow cytometry was adopted for analysis of cell cycle phase progression by PI staining method after serum starvation to synchroniz cells.
Results:(1)There were different expression levels between ROCK1 and ROCK2 in VSMCs, the expression of ROCK2 is higher eight times than that of ROCK1.(2)By using siRNA transfection, the expression of ROCK1 and ROCK2 were down regulated by 83.4% and 94.7% respectively as the cell migration experiments.(3)When reduced the expression of ROCK1, the migration of VSMCs was inhibited, but ROCK2 had no significant effect on VSMCs migration at the same condition. Also,ROCK activity blocked by Y-27632 also inhibited the VSMCs migration.(4)ROCK1 downregulation resulted in reduced levels of LIMK1 and LIMK2 phosphorylation, and ROCK2 had no effects on phosphorylation of LIMK1 and LIMK2. In addition, LIMK phosphorylation decreased after ROCK activity inhibition by Y-27632.(5)ROCK1 downregulation led to increased level of MLC phosphorylation, and ROCK2 had no significant effect on the level of MLC phosphorylation ROCK activity inhibition by Y-27632 also increased the level of MLC phosphorylation.(6)When ROCK1 and ROCK2 were down regulated respectively, no significant difference was observed in cell growth, and the cell cycle G1/S phase was also not altered obviously, whereas ROCK activity inhibition by Y-27632 leads to the inhibition of the G1/S phase progress.
Conclusions:(1)In the two ROCK informs, ROCK1, but not ROCK2, plays a leading role in VSMCs migration.(2)ROCK1 controls the migration of VSMCs via reducing the levels of LIMK phosphorylation and increasing MLC phosphorylation.(3)ROCK1 and ROCK2 have the same effects on the proliferation of VSMCs.
引文
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3. Paul Holvoet, Peter Sinnaeve, Leuven et al..Angio-associated migratory Cell Protein and Smooth Muscle Cell Migration in Development of Restenosis and Atherosclerosis. Journal of the American College of Cardiology. 2008, 52(4): 312-314.
4. Willis AI. Pierre-Paul D. Sumpio BE. Gahtan V. Vascular Smooth Muscle Cell Migration: Current Research and Clinical Implications Vascular and Endovascular Surgery, 2004, 38(1): 11-23.
5. Hedin U. Roy J. Tran PK.Control of smooth muscle cell proliferation in vascular disease. Current Opinion in Lipidology. 2004, 15(5): 559-65.
6. Song Lia James Jaehyun Moona Hui Miaob Gang Jinb Benjamin P.C. Chenb Suli Yuanb Yingli Hub Shunichi Usamib Shu Chienb. Signal Transduction in Matrix Contraction and the Migration of Vascular Smooth Muscle Cells in Three Dimension- al Matrix. J Vasc Res 2003, 40: 378–388.
7. Tammy M. Seasholtz, Mousumi Majumdar, Daniel D. Kaplan and Joan Heller Brown. Cell DNA Synthesis and Migration Rho and Rho Kinase Mediate Thrombin Stimulated Vascular Smooth Muscle.Circ. Res. 1999, 84: 1186-1193.
8. Daniel R. Croft and Michael F Olson. The Rho GTPase Effector ROCK Regulates Cyclin A, Cyclin D1,and p27Kip1 Levels by Distinct Mechanisms. MOLECULAR AND CELLULAR BIOLOGY, June 2006, 4612–4627.
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1. Astrid Trion, MSc, and Arnoud van der Laarse, Leiden, V.Vascular smooth muscle cells and calcification in Atherosclerosis . The Netherlands .American Heart Journal. 2004, 147(5): 808–814.
2. Vascular smooth muscle cell proliferation in the pathogenesis of atherosclerotic cardiovascular diseases. Histology & Histopathology. 2000, 15(2): 557-71.
3. Paul Holvoet, Peter Sinnaeve, Leuven et al..Angio-associated migratory Cell Protein and Smooth Muscle Cell Migration in Development of Restenosis and Atherosclerosis. Journal of the American College of Cardiology. 2008, 52(4): 312-314.
4. Willis AI. Pierre-Paul D. Sumpio BE. Gahtan V. Vascular Smooth Muscle Cell Migration: Current Research and Clinical Implications Vascular and Endovascular Surgery, 2004, 38(1): 11-23.
5. Hedin U. Roy J. Tran PK.Control of smooth muscle cell proliferation in vascular disease. Current Opinion in Lipidology. 2004, 15(5): 559-65.
6. Song Lia James Jaehyun Moona Hui Miaob Gang Jinb Benjamin P.C. Chenb Suli Yuanb Yingli Hub Shunichi Usamib Shu Chienb. Signal Transduction in Matrix Contraction and the Migration of Vascular Smooth Muscle Cells in Three Dimension- al Matrix. J Vasc Res 2003, 40: 378–388.
7. Tammy M. Seasholtz, Mousumi Majumdar, Daniel D. Kaplan and Joan Heller Brown. Cell DNA Synthesis and Migration Rho and Rho Kinase Mediate Thrombin Stimulated Vascular Smooth Muscle.Circ. Res. 1999, 84: 1186-1193.
8. Daniel R. Croft and Michael F Olson. The Rho GTPase Effector ROCK Regulates Cyclin A, Cyclin D1,and p27Kip1 Levels by Distinct Mechanisms. MOLECULAR AND CELLULAR BIOLOGY, June 2006, 4612–4627.
9. Ishizaki T, Maekawa M, Fujisawa K. small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 1996, 15: 1885–1893.
10. Matsui T, Amano M, Yamamoto et al. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 1996, 15: 2208–2216.
11. Leung T, Manser E, Tan et al. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes.J Biol Chem. 1995, 270: 29051–29054.
12. Kensuke Noma, Naotsugu Oyama and James K. Liao.Physiological role of ROCKs in the cardiovascular system.Am J Physiol Cell Physiol. 2006, 290: 661-668.
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15. Yuepeng Wang, Xiaoyu Rayne Zheng, Nadeene Riddick et al .ROCK Isoform Regulation of Myosin Phosphatase and Contractility in Vascular Smooth Muscle Cells. .Circulation Research. 2009, 104: 531-540.
16.Jawien A,Bowen-Pope DF,Lindner V, Schwartz SM, Clowes AW. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest. 1992, 89: 507–511.
17. Arber S, Barbayannis FA, Hanser H, et al: Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature. 1998; 393: 805-809.
18. Ohashi K, Nagata K, Maekawa M, Ishizaki T, Narumiya S, Mizuno K Rho-associated kinase ROCK activates LIMkinase 1 by phosphorylation at threonine 508 within the activation loop. J Biol Chem 2000, 275: 3577–3582
19. Amano T, Tanabe K, Eto T, Narumiya S, Mizuno K. LIM kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505. Biochem J. 2001, 354: 149–159
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