Bailleux Anne, Wendum Dominique, Audubert François, Jouniaux Anne-Marie, Koumanov Kamen, Trugnan Germain, Masliah Joëlle
INSERM Unité 538, Université Pierre et Marie Curie, CHU Saint-Antoine, 27 rue Chaligny, 75012 Paris, France.
Biochem J. 2004 Mar 1;378(Pt 2):307-15. doi: 10.1042/BJ20031014.
Madin-Darby canine kidney type II cells were shown to release low amounts of AA (arachidonic acid) and prostaglandin E2 in response to various stimuli when analysed after cell confluence. In contrast, non-confluent Madin-Darby canine kidney type II cells released much higher amounts of AA and prostaglandin E2. In both stationary and non-confluent cells, AA was released by type IV cPLA2 (cytosolic phospholipase A2), as shown by the use of specific inhibitors and by analysis of the profile of fatty acids released. This confluence-dependent cPLA2 activation was not due to a difference in expression, or in phosphorylation of the enzyme, or in the amount of its substrate. To find out the mechanism by which cPLA2 activation may be regulated as a function of cell confluence, immunofluorescence and co-immunoprecipitation experiments were performed using cPLA2, p11, a natural inhibitor of the enzyme, and annexin II, the natural ligand of p11. These three proteins were expressed at a constant level, regardless of the cell confluence. In contrast, whereas annexin II and cPLA2 interacted at a constant rate, p11 and cPLA2 interacted more strongly in stationary cells, thus indicating that cPLA2 activation is regulated by its accessibility to p11, independent of their expression level. Our results indicate that, in epithelial cells, the cell confluence, i.e. the establishment of cell-cell contacts, rather than cell proliferation directly controls cPLA2 activation by changing the stoichiometry of p11/cPLA2 interaction.
在细胞汇合后进行分析时，发现马-达二氏犬肾II型细胞在受到各种刺激时，释放的花生四烯酸（AA）和前列腺素E2的量较低。相比之下，未汇合的马-达二氏犬肾II型细胞释放的AA和前列腺素E2的量要高得多。在静止细胞和未汇合细胞中，IV型胞质磷脂酶A2（cPLA2）均可释放AA，这通过使用特异性抑制剂以及对释放的脂肪酸谱进行分析得以证明。这种依赖汇合的cPLA2激活并非由于该酶的表达、磷酸化或其底物量存在差异所致。为了找出cPLA2激活可能作为细胞汇合功能而受到调节的机制，使用cPLA2、p11（该酶的天然抑制剂）和膜联蛋白II（p11的天然配体）进行了免疫荧光和免疫共沉淀实验。这三种蛋白质的表达水平恒定，与细胞汇合情况无关。相反，虽然膜联蛋白II和cPLA2以恒定速率相互作用，但p11和cPLA2在静止细胞中的相互作用更强，这表明cPLA2的激活是由其与p11的可及性调节的，与它们的表达水平无关。我们的结果表明，在上皮细胞中，细胞汇合，即细胞间接触的建立，而非细胞增殖，通过改变p11/cPLA2相互作用的化学计量直接控制cPLA2的激活。
