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.
- annexin II
- epithelial cell
- phospholipase A2 (PLA2)
↵1 Present address: Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
Abbreviations used: AA, arachidonic acid; AACOCF3, arachidonyltrifluoromethyl ketone; Ab, antibody; AxII, annexin II; BEL (=HELSS), bromoenol lactone suicide substrate; Caco-2 cells, colon carcinoma-2 cells; FCS, foetal calf serum; mAb, monoclonal Ab; MDCK, Madin–Darby canine kidney; NEFA, non-esterified (‘free’) fatty acid; pAb, polyclonal Ab; PLA2, phospholipase A2; cPLA2, iPLA2, sPLA2, cytosolic, intracellular Ca2+-independent, secretory Ca2+-dependent PLA2 respectively.
- The Biochemical Society, London ©2004