Couturier C, Antonio V, Brouillet A, Béréziat G, Raymondjean M, Andréani M
Unité Propre de Recherche de l'Université Pierre et Marie Curie, associée au CNRS (ESA7079), Paris, France.
Arterioscler Thromb Vasc Biol. 2000 Dec;20(12):2559-65. doi: 10.1161/01.atv.20.12.2559.
Type II secreted phospholipase A(2) (sPLA(2)) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA(2), which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA(2) was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time- and concentration-dependent increase in sPLA(2) gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-ss and -delta antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA(2) promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA(2) mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-ss and -delta acting through a protein kinase A signaling pathway.
II型分泌型磷脂酶A2（sPLA(2)）可从磷脂中释放重要炎性脂质介质的前体。一些观察结果表明，sPLA(2)与诸如关节炎等慢性炎症性疾病有关，它在动脉壁动脉粥样硬化的形成中起作用。在对照血管平滑肌细胞（VSMC）中未检测到sPLA(2)。用增加细胞内cAMP的试剂（如福斯可林、二丁酰[db]-cAMP）处理VSMC，导致sPLA(2)基因表达呈时间和浓度依赖性增加。半定量逆转录聚合酶链反应（RT-PCR）显示蛋白激酶A抑制剂对福斯可林诱导的mRNA有明显的剂量依赖性抑制作用。用C/EBP共有寡核苷酸以及大鼠启动子的C/EBP寡核苷酸对经福斯可林处理和db-cAMP处理的VSMC的核蛋白进行电泳迁移率变动分析，结果显示与对照VSMC相比结合增强。用特异性蛋白激酶抑制剂H89孵育VSMC，也可阻断db-cAMP和福斯可林诱导的核C/EBP与大鼠启动子C/EBP位点的结合。使用CRE共有寡核苷酸时结合无变化。抗体显示形成了特异性的C/EBP/DNA复合物，其中大多数被C/EBP-ss和-delta抗体超迁移。荧光素酶报告基因测定证实了C/EBP的功能激活。包含来自大鼠sPLA(2)启动子的4个串联重复拷贝的C/EBP元件并与荧光素酶相连的构建体，通过与蛋白激酶A催化亚基的表达载体共转染在VSMC中被转录激活。在用福斯可林或db-cAMP处理的转染VSMC中它也被显著激活。H89抑制这种激活。因此我们得出结论，cAMP升高剂所产生的sPLA(2) mRNA和酶活性的增加是由一种涉及核C/EBP-ss和-delta通过蛋白激酶A信号通路起作用的机制所控制。
