溶血磷脂酰胆碱对血管平滑肌细胞的增殖作用及其信号通路
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摘要
目的研究溶血磷脂酰胆碱(LPC)对人血管平滑肌细胞的增殖作用,并阐明其受体介导信号转导机理。
内容培养的人大动脉平滑肌细胞,用LPA和LPC及LPA受体特异性拮抗剂Ki16425和Gi蛋白特异性抑制剂PTX刺激2-3d, MTT比色法观察AoSMCs增殖；实时荧光定量PCR分析LPA受体和Autotaxin的基因表达量；采用蛋白印迹技术检测血管平滑肌细胞在受到LPA、LPC、Ki16425和PTX刺激后,观察ERK1/2在磷酸化程度上的差别。
结果MTT法显示LPA、LPC均能促进AoSMCs增殖,且Ki16425和PTX均能抑制它们的增殖作用。荧光定量RT-PCR结果表明,在AoSMCs细胞中,LPA1-5五个受体均有表达,其中LPA1受体的表达量远远高于其它几个受体。Autotaxin基因在血管平滑肌细胞中大量表达,并且显示出很高的磷脂酶D活性,使LPC转化为LPA。Western blot结果表明,用LPA、LPC和PDGF单独刺激AoSMC细胞均能使ERK1/2信号通路活化。与Ki16425同时作用和PTX预处理AoSMC细胞时,LPA和LPC诱导的p-ERK1/2水平明显降低,而PDGF诱导的ERK1/2磷酸化水平未受影响。
结论1)研究首次发现,血管平滑肌细胞本身表达的Autotaxin参与由LPC刺激细胞的增殖作用。在细胞和细胞培养液中均检测到Autotaxin的蛋白表达和磷脂酶D的活性。2) LPC在胞外通过LPC-LPA1和LPC-LPA-LPA1两种途径激活血管平滑肌细胞LPA1受体,在血管平滑肌细胞内LPC促细胞增殖的信号转导途径是LPA1-Gi-ERK。3) ox-LDL及其中的LPC是引起动脉粥样硬化的重要因子,并控制血液中的ox-LDL浓度对预防和治疗动脉粥样硬化有重要的意义。
AIM:To study the effect of lysophosphatidylcholine (LPC) on proliferation of vascular smooth muscle cells (VSMCs) and it's signaling pathway involved in the proliferation.
Content:VSMCs were cultured and stimulated by LPA, and LPC at present with or without Ki16425 (An antagonist of LPA1 receptor) and PTX for 2 to 3 days, then their proliferation was measured by MTT assay. Gene expression of lysophospholipid receptors and autotaxin were determined by real-time quantitative PCR. Western blot assay was used to detect the ERK1/2 phosphorylation levels before and after the stimulation with LPA, at present with or without Ki 16425 and PTX.
Results:MTT assay showed that both the LPA and LPC stimulated VSMCs proliferation, which could be inhibited by Ki16425 or PTX. Real time quantitative PCR analysis showed that all five receptors of LPA are expressed in AoSMCs, among which the expression of LPA1 is far higher than others. The gene that encoding autotaxin is highly expressed in AoSMCs, and autotaxin has phospholipase D activity that hydrolyze LPC to LPA. Western blot analysis showed that LPA, LPC and PDGF can stimulate ERK phosphorylation all alone. Both the Ki 16425 and PTX decrease the expression of P-ERKI/2 induced by LPA and LPC, but not by PDGF.
Conclusion:1) In this study, we found that autotaxin expressed in the cells was related to the cell proliferation induced by LPC. And autotaxin have high phospholipase D activity.2) LPC can stimulate LPA1 receptor through both the LPC-LPA1 and LPC-LPA-LPA1 pathway, and induce cell proliferation through LPA1-Gi-ERK pathway in VSMCs.3) ox-LDL and its content LPC are important factors for induced atherosclerosis. Thus controlling the concentration of ox-LDL in blood is important in preventing and curing the atherosclerosis.
内容培养的人大动脉平滑肌细胞,用LPA和LPC及LPA受体特异性拮抗剂Ki16425和Gi蛋白特异性抑制剂PTX刺激2-3d, MTT比色法观察AoSMCs增殖；实时荧光定量PCR分析LPA受体和Autotaxin的基因表达量；采用蛋白印迹技术检测血管平滑肌细胞在受到LPA、LPC、Ki16425和PTX刺激后,观察ERK1/2在磷酸化程度上的差别。
结果MTT法显示LPA、LPC均能促进AoSMCs增殖,且Ki16425和PTX均能抑制它们的增殖作用。荧光定量RT-PCR结果表明,在AoSMCs细胞中,LPA1-5五个受体均有表达,其中LPA1受体的表达量远远高于其它几个受体。Autotaxin基因在血管平滑肌细胞中大量表达,并且显示出很高的磷脂酶D活性,使LPC转化为LPA。Western blot结果表明,用LPA、LPC和PDGF单独刺激AoSMC细胞均能使ERK1/2信号通路活化。与Ki16425同时作用和PTX预处理AoSMC细胞时,LPA和LPC诱导的p-ERK1/2水平明显降低,而PDGF诱导的ERK1/2磷酸化水平未受影响。
结论1)研究首次发现,血管平滑肌细胞本身表达的Autotaxin参与由LPC刺激细胞的增殖作用。在细胞和细胞培养液中均检测到Autotaxin的蛋白表达和磷脂酶D的活性。2) LPC在胞外通过LPC-LPA1和LPC-LPA-LPA1两种途径激活血管平滑肌细胞LPA1受体,在血管平滑肌细胞内LPC促细胞增殖的信号转导途径是LPA1-Gi-ERK。3) ox-LDL及其中的LPC是引起动脉粥样硬化的重要因子,并控制血液中的ox-LDL浓度对预防和治疗动脉粥样硬化有重要的意义。
AIM:To study the effect of lysophosphatidylcholine (LPC) on proliferation of vascular smooth muscle cells (VSMCs) and it's signaling pathway involved in the proliferation.
Content:VSMCs were cultured and stimulated by LPA, and LPC at present with or without Ki16425 (An antagonist of LPA1 receptor) and PTX for 2 to 3 days, then their proliferation was measured by MTT assay. Gene expression of lysophospholipid receptors and autotaxin were determined by real-time quantitative PCR. Western blot assay was used to detect the ERK1/2 phosphorylation levels before and after the stimulation with LPA, at present with or without Ki 16425 and PTX.
Results:MTT assay showed that both the LPA and LPC stimulated VSMCs proliferation, which could be inhibited by Ki16425 or PTX. Real time quantitative PCR analysis showed that all five receptors of LPA are expressed in AoSMCs, among which the expression of LPA1 is far higher than others. The gene that encoding autotaxin is highly expressed in AoSMCs, and autotaxin has phospholipase D activity that hydrolyze LPC to LPA. Western blot analysis showed that LPA, LPC and PDGF can stimulate ERK phosphorylation all alone. Both the Ki 16425 and PTX decrease the expression of P-ERKI/2 induced by LPA and LPC, but not by PDGF.
Conclusion:1) In this study, we found that autotaxin expressed in the cells was related to the cell proliferation induced by LPC. And autotaxin have high phospholipase D activity.2) LPC can stimulate LPA1 receptor through both the LPC-LPA1 and LPC-LPA-LPA1 pathway, and induce cell proliferation through LPA1-Gi-ERK pathway in VSMCs.3) ox-LDL and its content LPC are important factors for induced atherosclerosis. Thus controlling the concentration of ox-LDL in blood is important in preventing and curing the atherosclerosis.
引文
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41. Ludwig, M.G., M. Vanek, D. Guerini, etc. Proton-sensing G-protein-coupled receptors. Nature, 2003,425(6953):93-8.
42. Wang, J.Q., J. Kon, C. Mogi, etc. TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor. J Biol Chem,2004,279(44):45626-33.
43. Tomura, H., C. Mogi, K. Sato, etc. Proton-sensing and lysolipid-sensitive G-protein-coupled receptors:a novel type of multi-functional receptors. Cell Signal,2005,17(12):1466-76.
44. Tomura, H., J.Q. Wang, M. Komachi, etc. Prostaglandin 1(2) production and cAMP accumulation in response to acidic extracellular pH through OGRl in human aortic smooth muscle cells. J Biol Chem,2005,280(41):34458-64.
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21. Bandoh, K., J. Aoki, H. Hosono, etc. Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid. J Biol Chem,1999, 274(39):27776-85.
22. Contos, J.J., I. Ishii and J. Chun. Lysophosphatidic acid receptors. Mol Pharmacol,2000, 58(6):1188-96.
23. Okajima, F. and Y. Kondo. Pertussis toxin inhibits phospholipase C activation and Ca2+ mobilization by sphingosylphosphorylcholine and galactosylsphingosine in HL60 leukemia cells. Implications of GTP-binding protein-coupled receptors for lysosphingolipids. J Biol Chem,1995,270(44):26332-40.
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50. 邓小燕,王贵学等.动脉系统中致动脉粥样性脂质的浓度极化现象.中国科学C辑,2002,32：559-567.
51. Farmakis, T.M., J.V. Soulis, G.D. Giannoglou, etc. Wall shear stress gradient topography in the normal left coronary arterial tree:possible implications for atherogenesis. Curr Med Res Opin, 2004,20(5):587-96.
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53. Karliner, J.S. Lysophospholipids and the cardiovascular system. Biochim Biophys Acta,2002, 1582(1-3):216-21.
54. Chen, X., X.Y. Yang, N.D. Wang, etc. Serum lysophosphatidic acid concentrations measured by dot immunogold filtration assay in patients with acute myocardial infarction. Scand J Clin Lab Invest,2003,63(7-8):497-503.
55. Kume, N., M.I. Cybulsky and M.A. Gimbrone, Jr. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest,1992,90(3):1138-44.
56. 杨丽丽,凌文华.氧化型低密度脂蛋白与动脉粥样硬化国外医学内科学分册,27.
57. Aiyar, N., J. Disa, Z. Ao, etc. Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells. Mol Cell Biochem,2007,295(1-2):113-20.