Ligation of the α2M* Signaling Receptor Regulates Synthesis of Cytosolic Phospholipase A2

We have studied the regulation of cytosolic phospholipase A2 (cPLA2) synthesis in macrophages stimulated with receptor-recognized forms of α2-macroglobulin (α2M*). [35S]methionine-labeled cells were stimulated with α2M* and [35S]cPLA2 was immunoprecipitated with a monoclonal antibody directed against cPLA2. The precipitates were electrophoresed, immunoblotted, cPLA2 detected by Enhanced Chemifluorescence, and its radioactivity determined. Stimulation of cells with α2M* caused a two- to threefold increase in cPLA2 synthesis compared to buffer-treated cells which was consistently maximal at 200 pM of α2M*. Actinomycin D or cycloheximide treatment of cells drastically reduced α2M*-induced cPLA2 synthesis. Likewise, inhibition of protein kinase C with chelerythrin, farnesyl transferase with manumycin A, MEK kinase with U0126, Erk1/2 kinases with PD98059, p38MAPK with SB203580, PI 3-kinase with wortmannin or LY294002, p70s6k with rapamycin, or depletion of [Ca2+]i with either BAPTA/AM or EGTA drastically reduced α2M* induction of cPLA2. Inhibition of NFκB activation with BAY11-7182 or PGA1 also abolished α2M* induction of cPLA2. We conclude that α2M*-induced cPLA2 synthesis is controlled by [Ca2+]i levels, tyrosine kinase activity, the p21ras-dependent MAPK and PI 3-kinase downstream signaling pathways, and regulation of NFκB.