Posmantur R, Wang K K, Gilbertsen R B
Parke-Davis Pharmaceutical Research, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan, 48105, USA.
Exp Cell Res. 1998 Oct 10;244(1):302-9. doi: 10.1006/excr.1998.4214.
The caspase family of proteases has previously been implicated in the biochemical cascade leading to apoptotic cell death. Recently caspase-3 was reported to be cleaved into its catalytically active subunits (17 and 13 kDa) following phytohemagglutinin (PHA) activation of peripheral blood mononuclear cells (C. Miossec et al., J. Biol. Chem. 272, 13459-13462). More recently, J. M. Zapata and colleagues (J. Biol. Chem. 273, 6916-6920, 1998), however, proposed that caspase-3 activity detected during T-cell activation was due to a methodological artifact related to the composition of the cell lysis buffer. Here we show that in PHA-activated Jurkat T-cells using the recommended lysis buffer detailed by Zapata et al., a caspase-3-like protease is activated and is accompanied by cleavage of PARP and alpha-spectrin into cleavage products suggestive of caspase-3 proteolytic activation. LDH release did not increase following PHA stimulation in this paradigm. Two caspase inhibitors, carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) and acetyl-Asp-Glu-Val-Asp-CHO, blocked IL-2 release in a dose-dependent manner. Caspase-3-like protease-generated PARP and alpha-spectrin breakdown product formation was also reduced by Z-D-DCB. In addition, Jurkat T-cells costimulated with anti-CD3 plus anti-CD28 produced significant levels of IL-2 that were also blocked by these caspase inhibitors. Importantly, IL-2 was determined in cell culture supernatants, thus avoiding a cell lysis step that might have enabled activation of caspase-3 by granzyme B. Collectively, these data support the role of caspase-3-like protease activity in Jurkat T-cell activation and demonstrate that caspase-3 like activity is necessary for IL-2 release in PHA-activated and anti-CD3/anti-CD28 costimulated Jurkat T-cells.
蛋白酶的半胱天冬酶家族先前已被认为参与导致凋亡性细胞死亡的生化级联反应。最近有报道称，在植物血凝素（PHA）激活外周血单核细胞后，半胱天冬酶-3被切割成其具有催化活性的亚基（17 kDa和13 kDa）（C. Miossec等人，《生物化学杂志》272, 13459 - 13462）。然而，最近J. M. Zapata及其同事（《生物化学杂志》273, 6916 - 6920, 1998）提出，在T细胞激活过程中检测到的半胱天冬酶-3活性是由于与细胞裂解缓冲液组成相关的方法学假象。在此我们表明，在使用Zapata等人详细推荐的裂解缓冲液的PHA激活的Jurkat T细胞中，一种类似半胱天冬酶-3的蛋白酶被激活，并伴随着聚（ADP-核糖）聚合酶（PARP）和α-血影蛋白被切割成提示半胱天冬酶-3蛋白水解激活的切割产物。在此模式下，PHA刺激后乳酸脱氢酶（LDH）释放并未增加。两种半胱天冬酶抑制剂，苄氧羰基-天冬氨酸-CH2OC(O)-2,6-二氯苯（Z-D-DCB）和乙酰基-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-醛（Ac-DEVD-CHO）以剂量依赖方式阻断白细胞介素-2（IL-2）释放。Z-D-DCB也减少了类似半胱天冬酶-3的蛋白酶产生的PARP和α-血影蛋白降解产物的形成。此外，用抗CD3加抗CD28共刺激的Jurkat T细胞产生了显著水平的IL-2，这些半胱天冬酶抑制剂也能阻断其产生。重要的是，IL-2是在细胞培养上清液中测定的，从而避免了可能通过颗粒酶B激活半胱天冬酶-3的细胞裂解步骤。总体而言，这些数据支持类似半胱天冬酶-3的蛋白酶活性在Jurkat T细胞激活中的作用，并证明类似半胱天冬酶-3的活性对于PHA激活的和抗CD3/抗CD28共刺激的Jurkat T细胞中IL-2的释放是必需的。
