Altman A, Mally M I, Isakov N
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, California 92037.
Immunology. 1992 Jul;76(3):465-71.
Studies described herein were designed to examine the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA), and a Ca2+ ionophore (ionomycin), singly or in combination, on the activation and expression of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes (alpha, beta and gamma) at the protein and messenger RNA (mRNA) levels in T cells. These two agents induce the activation and proliferation of T lymphocytes by mimicking the action of inositol phospholipid-derived second messengers normally generated by triggering of the antigen-specific T-cell receptor (TcR)/CD3 complex. TPA-induced T-cell proliferation, expression of interleukin-2 receptor-alpha subunit (IL-2R alpha) and transferrin receptor, CD3 down-regulation and, lastly, the cytosol-to-membrane PKC translocation (determined by an enzymatic assay or by immunoblotting with a cross-reactive anti-PKC peptide antibody) were all facilitated by ionomycin. Immunoblots with isoenzyme-specific anti-PKC monoclonal antibodies demonstrated expression of immunoreactive PKC alpha, PKC beta and PKC gamma proteins that were translocated to the membrane upon TPA plus ionomycin stimulation. Resting T cells expressed abundant levels of mRNA for PKC alpha and PKC beta, but very low levels (relative to brain) of PKC gamma. TPA increased by two- to threefold the expression of PKC beta, but not of PKC alpha or PKC gamma, mRNA within 12 hr of stimulation. Ionomycin synergized with TPA in increasing the expression of PKC alpha and PKC beta mRNA. The two agents also synergized in inducing expression of additional activation/growth-associated genes, namely the c-myc protooncogene, ornithine decarboxylase (ODC) and IL-2R alpha. Ionomycin alone was inactive (or marginally active) in all of these assays. The translocation of distinct Ca(2+)-dependent PKC isoenzymes to the membrane and the up-regulation of PKC alpha and beta mRNA suggest that at least these two isoenzymes are involved in discrete steps of the pathway leading to T-cell activation and proliferation. Moreover, the combined effects of TPA and ionomycin on T-cell function and cell-surface antigen expression appear to be due, at least in part, to their synergistic activation of distinct PKC isoenzyme(s).
本文所述研究旨在考察十四酰佛波醇-13-乙酸酯（TPA）和钙离子载体（离子霉素）单独或联合作用，对T细胞中钙离子依赖性蛋白激酶C（PKC）同工酶（α、β和γ）在蛋白和信使核糖核酸（mRNA）水平的激活及表达的影响。这两种试剂通过模拟通常由抗原特异性T细胞受体（TcR）/CD3复合物触发产生的肌醇磷脂衍生的第二信使的作用，诱导T淋巴细胞的激活和增殖。离子霉素促进了TPA诱导的T细胞增殖、白细胞介素-2受体α亚基（IL-2Rα）和转铁蛋白受体的表达、CD3下调，以及最后胞质溶胶到膜的PKC易位（通过酶促测定或用交叉反应性抗PKC肽抗体进行免疫印迹法测定）。用同工酶特异性抗PKC单克隆抗体进行的免疫印迹显示，免疫反应性PKCα、PKCβ和PKCγ蛋白的表达，在TPA加离子霉素刺激后易位到膜上。静息T细胞表达大量的PKCα和PKCβ mRNA，但PKCγ的水平非常低（相对于脑）。TPA在刺激1２小时内使PKCβ的mRNA表达增加两到三倍，但不影响PKCα或PKCγ的mRNA表达。离子霉素与TPA协同增加PKCα和PKCβ mRNA的表达。这两种试剂在诱导其他激活/生长相关基因即c-myc原癌基因、鸟氨酸脱羧酶（ODC）和IL-2Rα的表达方面也具有协同作用。单独的离子霉素在所有这些测定中均无活性（或活性微弱）。不同的钙离子依赖性PKC同工酶向膜的易位以及PKCα和β mRNA的上调表明，至少这两种同工酶参与了导致T细胞激活和增殖途径的不同步骤。此外，TPA和离子霉素对T细胞功能和细胞表面抗原表达的联合作用似乎至少部分归因于它们对不同PKC同工酶的协同激活。
