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U937 cells deprived of endogenous annexin 1 demonstrate an increased PLA 2 activity
Author(s) -
Solito Egle,
RaguenesNicol Celine,
De Coupade Catherine,
BisagniFaure Anne,
RussoMarie Françoise
Publication year - 1998
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0701991
Subject(s) - transfection , clone (java method) , microbiology and biotechnology , sense (electronics) , phospholipase a2 , phorbol , biology , annexin , flow cytometry , cell culture , endogeny , western blot , phosphorylation , phospholipase a , chemistry , enzyme , protein kinase c , biochemistry , gene , genetics
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 A 2 (PLA 2 ) activity, U 937 cells were stably transfected both with a Sense and Antisense cDNA for An 1. PLA 2 activity was measured by Flow cytometry analysis utilizing the bis‐Bodipy‐C 11 ‐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 PLA 2 activity than the control Sense clone (15 S). Since the fluorescent probe measures the total PLA 2 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 PLA 2 involved (namely arachidonyl trifluoromethyl ketone or AACOCF3, which is specific for the cytosolic PLA 2 , and SB 203347 specific for the secretory PLA 2 ). 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 cPLA 2 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 PLA 2 .British Journal of Pharmacology (1998) 124 , 1675–1683; doi: 10.1038/sj.bjp.0701991