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

Abstract

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.

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

“…The mechanism underlying this effect is not clear, although the results obtained with DTT and BEL indicate that BEL-sensitive iPLA 2 activity may be responsible for the impairment of AA release, whereas cPLA 2 activity was present in both nondifferentiated and differentiated Caco-2 cultures. In contrast, Bailleux et al (38) reported that the postconfluence decrease in [ 3 H]AA release depends on cPLA 2 activity in the case of Madin-Darby canine kidney type II and Caco-2 cultures.…”

Cell culture models demonstrate that CFTR dysfunction leads to defective fatty acid composition and metabolism

“…Similarly, enzymes regulating fatty acid utilization are affected, based on two recent reports. Bailleux et al (22) observed that the attainment of confluency with the establishment of cell-to-cell contacts controlled the activation of the cytosolic form of phospholipase A 2 . This was a result of a stoichiometric change in the interaction of the phospholipase A 2 with the protein p11.…”

“…For experiments described thus far, cells were harvested at 24-48 h after 100% confluence. Because the degree of cell contact can affect fatty acid metabolism (22,23), the effect of cell density on fatty acid levels in 16HBE cells was examined (Fig. 4).…”

Cytosolic phospholipase A2 regulation in the hibernating thirteen-lined ground squirrel

“…Phosphorylation activates the enzyme but a rise in intracellular Ca 2+ , acting through the calcium-lipid binding domain, is needed to cause translocation of cPLA 2 from the cytosol to the membrane where it gains access to its substrate. However, recent studies have indicated that cPLA 2 interaction with other membrane lipids such as cholesterol, phosphatidylinositol bisphosphate, and ceramides, as well as interaction with proteins like annexins, p11 and vimentin, are needed for the complete activity of cPLA 2 [26,27]. Hence, the full story of the mechanisms of regulation of this enzyme during hibernation remains to be be determined as do the full range of metabolic consequences of cPLA 2 suppression.…”