U937 cells deprived of endogenous annexin 1 demonstrate an increased PLA2 activity.
Annexin 1 (An 1), a phospholipid and calcium binding protein, is strongly expressed in differentiated U 937 cells. In attempting to correlate the expression of An 1 with phospholipase A2 (PLA2) activity, U 937 cells were stably transfected both with a Sense and Antisense cDNA for An 1. PLA2 activity was measured by Flow cytometry analysis utilizing the bis-Bodipy-C11-PC fluorescent probe. U 937 cells stably transfected with the sense or antisense vectors were differentiated for 24 h with phorbol 12-myristate 13-acetate (PMA, 6 ng ml(-1)). Both in undifferentiated and differentiated cells, the Antisense clone (36.4 AS) showed consistently higher PLA2 activity than the control Sense clone (15 S). Since the fluorescent probe measures the total PLA2 activity, we used two different stimuli, PMA: (100 ng ml(-1)) or lipopolysaccharide (LPS, 10 ng ml(-1)), and two different inhibitors, to discriminate the PLA2 involved (namely arachidonyl trifluoromethyl ketone or AACOCF3, which is specific for the cytosolic PLA2, and SB 203347 specific for the secretory PLA2). In the Antisense clone the inhibitory effect of AACOCF was stronger [68%, P<0.025] than in the Sense, which may reflect the lower endogenous level of An 1 present in the cells. On the contrary, the inhibitory effect of SB 203347 [60% of inhibition] was identical in both clones. Since cPLA2 activity is correlated with its phosphorylation, Western and shift blot analysis were performed. They did not show any significative difference between the phosphorylated and non phosphorylated form of the enzyme in both the differentiated or not, Sense and Antisense clones. Furthermore the tyrosine phosphorylation analysis of An 1 showed that less than 10% of An 1 was phosphorylated irrespective of PMA presence or absence. From the pattern of inhibition observed, we propose that the endogenous unphosphorylated form of An 1 may act intracellularly to block the activity of a cytosolic PLA2.
Institut Cochin de Génétique Moléculaire Inserm U-332, Paris, France.
E
E
Solito
C
C
Raguenes-Nicol
C
C
de Coupade
A
A
Bisagni-Faure
F
F
Russo-Marie
D017305:Annexin A1 / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology*; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D008070:Lipopolysaccharides / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020298:U937 Cells
D017305:Annexin A1; D002135:Calcium-Binding Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate