Inhibitory effect of peptides derived from the N‐terminus of lipocortin 1 on arachidonic acid release and proliferation in the A549 cell line: identification of E‐Q‐E‐Y‐V as a crucial component

1 The ability of the glucocorticoid‐induced protein lipocortin 1 (LC1) to inhibit arachidonic acid release and cell proliferation in A549 cells may be mimicked by a sequence taken from the N‐terminal, LC1 13–25 (FIENEEQEYVQTV). We have now synthesized and tested for biological activity a library of 25 smaller peptides derived from this sequence. 2 Peptides were tested in two assays: A549 cells were prelabelled with tritiated arachidonic acid and thapsigargin (50 n M ) and EGF (10 n M ) used to stimulate the release of this fatty acid. Cell proliferation was determined by counting cell numbers following 3 day incubation with these peptides, or controls. 3 Many of the peptides were highly insoluble but could be more readily dissolved in aqueous solution in the presence of commercial liposomes or phosphatidyl serine (5 μ M ). Since neither of these agents alone had any effect on arachidonic acid release or cell proliferation, all peptides were tested in the presence of 5 μ M phosphatidyl serine. Under these conditions LC1 13–25 was active in both assay systems with an IC 40 of 40.7 and 57.0 μ M respectively. 4 Deletion of amino acids from the C‐terminus of the peptide progressively diminished (2–3 fold) the molar potency of LC1 13–25 in both assays: after the removal of Val 22 biological activity was virtually undetectable or very weak (<30% of LC1 13–25 ). 5 Removal of amino acids from the N‐terminus also lead to a progressive reduction (3–5 fold) in the molar potency of the peptides and biological activity became undetectable, or very weak, after the removal of Glu 18 . 6 All active peptides contained the core sequence EQEYV(Glu‐Gln‐Glu‐Tyr‐Val) which seems to represent a crucial component of the pharmacophore, although this sequence on its own was inactive and the shortest peptide with significant activity was LC1 18–25 (EQEYVQTV). 7 Methoxylation of Tyr 21 abolished the ability of LC1 18–25 to inhibit cell proliferation and arachidonic acid release. A cyclized version of LC1 18–25 was also tested and found to be inactive. 8 LC1 18–25 (178 μ M ) inhibits cPLA 2 activation in A549 cells as judged by a band‐shift assay, whereas equimolar concentrations of an inactive peptide LC1 19–25 were without effect in this assay system. 9 Several possible mechanisms whereby these peptides act are discussed in the light of LC1 biology and of the effect of glucocorticoids on cell function.British Journal of Pharmacology (1998) 123 , 975–983; doi: 10.1038/sj.bjp.0701679