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 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.
INSERM Unité 538, Université Pierre et Marie Curie, CHU Saint-Antoine, 27 rue Chaligny, 75012 Paris, France.
Anne
A
Bailleux
Dominique
D
Wendum
François
F
Audubert
Anne-Marie
AM
Jouniaux
Kamen
K
Koumanov
Germain
G
Trugnan
Joëlle
J
Masliah
D000818:Animals; D017306:Annexin A2 / Q000378:metabolism*; D016718:Arachidonic Acid / Q000032:analysis / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D004285:Dogs; D015536:Down-Regulation; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D054513:Group IV Phospholipases A2; D006801:Humans; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D009418:S100 Proteins / Q000378:metabolism*; D000099248:S100 Calcium Binding Protein A10
D017306:Annexin A2; D016718:Arachidonic Acid; D054513:Group IV Phospholipases A2; D010741:Phospholipases A; D054467:Phospholipases A2; D009418:S100 Proteins; D000099248:S100 Calcium Binding Protein A10; C516389:PLA2G4A protein, human