Cytosolic phospholipase A2-p11 interaction controls arachidonic acid release as a function of epithelial cell confluence

Madin-Darby canine kidney type II cells were shown to release low amounts of AA (arachidonic acid) and prostaglandin E-2 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 prostarglandin E-2. In both stationary and non-confluent cells, AA was released by type IV cPLA(2) (cytosolic phospholipase A(2)), as shown by the use of specific inhibitors and by analysis of the profile of fatty acids released. This confluence-dependent cPLA(2) 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 cPLA(2) activation may be regulated as a function of cell confluence, immunofluorescence and co-immunoprecipitation experiments were performed using cPLA(2), 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 11 and cPLA(2) interacted at a constant rate, p11 and cPLA(2) interacted more strongly in stationary cells, thus indicating that cPLA(2) 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' cPLA(2) activation by changing the stoichiometry of p11/cPLA(2) interaction.