Abstract
A colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell line (BAC1.2F5) and peritoneal macrophages were used to investigate the relationship between growth factor-dependent phosphorylation/activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) and arachidonic acid metabolism. The addition of CSF-1 to quiescent BAC1.2F5 cells was followed by the rapid phosphorylation, electrophoretic gel retardation, and stable increase in the specific activity of cPLA2 that correlated with the activation of ERK kinases. cPLA2 phosphorylation depended on the presence of growth factor and persisted throughout the cell cycle. CSF-1 inhibited prostaglandin E2 production and did not enhance arachidonic acid release or increase the levels of lysophosphatidylcholine or glycerophosphocholine. Treatment of BAC1.2F5 cells with the calcium ionophore A23187 plus CSF-1 did not stimulate eicosanoid release. Instead, CSF-1 enhanced the rate of exogenous arachidonic acid incorporation into phosphatidylcholine and its subsequent transfer to phosphatidylethanolamine suggesting that higher rates of arachidonic acid acylation may contribute to the suppression of prostaglandin production. In peritoneal macrophages, ERK kinase activity was stimulated and cPLA2 was phosphorylated and activated in response to CSF-1. However, CSF-1 did not trigger eicosanoid release but did augment arachidonic acid mobilization and prostaglandin E2 production elicited by zymosan and A23187. Thus, cPLA2 phosphorylation/activation and calcium mobilization are not the only determinants for eicosanoid release, and additional components in differentiated tissue macrophages are also required.
Highlights
A colony-stimulating factor1 (CSF-Wdependent mu- generation of eicosanoid products associated with a broadspecrinemacrophage cell line (BAC1.2F5)andperitoneal trum of physiological responses
CSF-1did dues resultingin a stable increase incPLA, specific activity of not trigger eicosanoid release but did augment arachi- about 2-fold in cell extracts (20-25). cPLA,phosphorylation donic acid mobilization and prostaglandin E, produc- correlates with the appearanocfea more slowly migrating election elicited byzymosanandA23187T. husc,PLA, trophoretic form suggesting that agonist-stimulated phosphophosphorylatiodactivation andcalciummobilization rylation is stoichiometric (22, 23, 25)
Our data show that CSF-1-dependent phosphorylation and activation of the 85 kDa cPLA, is common to BAC1.2F5 cells and peritoneal macrophages;there are significant differences in theregulation of arachidonic acid metabolism in the two cell systems
Summary
To measure the incorporation of phosphate into cPLA,,100-mm dishes of BAC1.2F5 cells were deprivedof CSF-1for 18h in phosphate-. After centrifugadishes of cells werelabeled with 0.25 pCi/mlof r3H1arachidonic acid for tion at 16,000 x g for 10 min, the supernatant was assayed for ERK the first 14 h of CSF-1 deprivation, and the media were removed kinase activity in a reaction mixture containing 50 mM p-glycerophosand the cells werewashed to remove exogenouslabel and incubated for phate, pH 7.2,lO mM MgCl,, 100~ M A T(P1pCi of [y-32PlATP/assay), 25 an additional 4 h in 8 ml of DMEM plus 15%FCS, 1%glutamine, and pg/ml IP-20,l mM EGTA, 200w EGFRS62-68a1n,d 20 pl of celllysate The cells were washed with PBS, pg of protein) in a final volume of 40 pl and incubated for 10 min at and the media werereplaced with DMEM plus BSA either with or 30 "C.The reactions were stopped with 10 pl of 25%trichloroaceticacid without 90 ng/ml recombinant CSF-1. The me- and were spotted onto Whatman phosphocellulose filter discs cPLA 2
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