Pardo Julián, Buferne Michel, Martínez-Lorenzo María-José, Naval Javier, Schmitt-Verhulst Anne-Marie, Boyer Claude, Anel Alberto
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Int Immunol. 2003 Dec;15(12):1441-50. doi: 10.1093/intimm/dxg141.
CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.
CD8(+) 细胞毒性T淋巴细胞（CTL）克隆能够发挥穿孔素依赖性和Fas依赖性细胞毒性作用。我们在本研究中表明，磷脂酰肌醇3激酶（PI3K）抑制剂渥曼青霉素和LY294002可阻止TCR/CD3诱导的功能性Fas配体（FasL）表达，但不影响穿孔素依赖性细胞毒性。经典蛋白激酶C（PKC）亚型的特异性抑制剂Gö6976完全抑制穿孔素依赖性细胞毒性，仅轻微影响TCR/CD3诱导的FasL表达，而使用对PKCθ具有更高特异性的抑制剂罗特勒素时则观察到相反的结果。为了进一步研究FasL表达对PI3K的依赖性，使用了由FasL启动子控制的荧光素酶报告基因。在用显性负性PI3K（PI3K-DN）转染的细胞中，抗CD3单克隆抗体诱导的报告基因诱导被消除，而在用组成型活性PI3K（PI3K*）转染的细胞中则增加。用PKCε尤其是PKCθ的组成型活性突变体（A/E）转染可改善抗CD3单克隆抗体诱导的报告基因表达，并完全消除渥曼青霉素的抑制作用，而用显性负性（K/R）PKCθ转染则可阻止报告基因的诱导。最后，用PKCα A/E而非PKCθ A/E转染与离子霉素协同作用，可诱导CTL系1.3E6SN脱颗粒。总之，结果表明TCR/CD3诱导的FasL基因转录受PI3K和PKCθ激活的控制，而该信号通路与CTL脱颗粒无关，CTL脱颗粒更依赖于经典PKC亚型的激活。
