Misra U K, Pizzo S V
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Arch Biochem Biophys. 2001 Feb 15;386(2):227-32. doi: 10.1006/abbi.2000.2199.
We have studied the regulation of cytosolic phospholipase A2 (cPLA2) synthesis in macrophages stimulated with receptor-recognized forms of alpha2-macroglobulin (alpha2M*). [35S]methionine-labeled cells were stimulated with alpha2M* 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 alpha2M* caused a two- to threefold increase in cPLA2 synthesis compared to buffer-treated cells which was consistently maximal at 200 pM of alpha2M*. Actinomycin D or cycloheximide treatment of cells drastically reduced alpha2M*-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 alpha2M* induction of cPLA2. Inhibition of NFKB activation with BAY11-7182 or PGA1 also abolished alpha2M* induction of cPLA2. We conclude that alpha2M*-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 NFkappaB.
我们研究了用受体识别形式的α2-巨球蛋白（α2M*）刺激巨噬细胞时胞质磷脂酶A2（cPLA2）合成的调控。用α2M刺激[35S]甲硫氨酸标记的细胞，并用针对cPLA2的单克隆抗体免疫沉淀[35S]cPLA2。将沉淀物进行电泳、免疫印迹，通过增强化学荧光检测cPLA2，并测定其放射性。与用缓冲液处理的细胞相比，用α2M刺激细胞导致cPLA2合成增加两到三倍，在200 pM的α2M时始终达到最大值。用放线菌素D或环己酰亚胺处理细胞可显著降低α2M诱导的cPLA2合成。同样，用白屈菜红碱抑制蛋白激酶C、用曼诺霉素A抑制法尼基转移酶、用U0126抑制MEK激酶、用PD98059抑制Erk1/2激酶、用SB203580抑制p38MAPK、用渥曼青霉素或LY294002抑制PI 3-激酶、用雷帕霉素抑制p70s6k，或用BAPTA/AM或EGTA耗尽细胞内[Ca2+]i，均可显著降低α2M对cPLA2的诱导。用BAY11-7182或PGA1抑制NFκB激活也可消除α2M对cPLA2的诱导。我们得出结论，α2M*诱导的cPLA2合成受细胞内[Ca2+]i水平、酪氨酸激酶活性、p21ras依赖性MAPK和PI 3-激酶下游信号通路以及NFκB调控。
