Department of Infection Biology and Research Institute for Medical Sciences, Chungnam National University School of Medicine, Daejeon 301-131, South Korea.
Exp Parasitol. 2013 Apr;133(4):462-71. doi: 10.1016/j.exppara.2013.01.005. Epub 2013 Jan 16.
Toxoplasma gondii-infected cells are resistant to various apoptotic stimuli, however, the role of the pro-apoptotic BH3-only Bad protein in T. gondii-imposed inhibition of host cell apoptosis in connection with the phosphoinositide 3-kinase (PI3K)-PKB/Akt pathway was not well delineated. Here, we investigated the signaling patterns of Bad, Bax and PKB/Akt in T. gondii-infected and uninfected THP-1 cells treated with staurosporine (STS) or PI3K inhibitors. STS treatment, without T. gondii infection, reduced the viability of THP-1 cells in proportion to STS concentration and triggered many cellular death events such as caspase-3 and -9 activation, Bax translocation, cytochrome c release from host cell mitochondria into cytosol, and PARP cleavage in the host cell. However, T. gondii infection eliminated the STS-triggered mitochondrial apoptotic events described above. Additionally, T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bad in a parasite-load-dependent manner which subsequently inhibited Bax translocation. The PI3K inhibitors, LY294002 and Wortmannin, both blocked parasite-induced phosphorylation of PKB/Akt and Bad. Furthermore, THP-1 cells pretreated with these PI3K inhibitors showed reduced phosphorylation of Bad in a dose-dependent manner and subsequently failed to inhibit the Bax translocation, also these cells also failed to overcome the T. gondii-imposed inhibition of host cell apoptosis. These data demonstrate that the PI3K-PKB/Akt pathway may be one of the major route for T. gondii in the prevention of host cell apoptosis and T. gondii phosphorylates the pro-apoptotic Bad protein to prevent apoptosis.
刚地弓形虫感染的细胞对各种凋亡刺激物具有抗性，然而，促凋亡 BH3 仅 Bad 蛋白在刚地弓形虫抑制宿主细胞凋亡过程中的作用，与磷酸肌醇 3-激酶（PI3K）-蛋白激酶 B/akt 途径有关，这一作用尚未得到很好的描述。在这里，我们研究了 Bad、Bax 和 PKB/Akt 在刚地弓形虫感染和未感染 THP-1 细胞中用星形孢菌素（STS）或 PI3K 抑制剂处理后的信号转导模式。STS 处理（无刚地弓形虫感染）使 THP-1 细胞的活力与 STS 浓度成比例下降，并触发许多细胞死亡事件，如 caspase-3 和 -9 激活、Bax 易位、宿主细胞线粒体中的细胞色素 c 释放到细胞质中，以及宿主细胞中 PARP 的裂解。然而，刚地弓形虫感染消除了上述 STS 触发的线粒体凋亡事件。此外，刚地弓形虫在体外和体内感染以寄生虫负荷依赖的方式诱导 PKB/Akt 和 Bad 的磷酸化，随后抑制 Bax 易位。PI3K 抑制剂 LY294002 和 Wortmannin 均可阻断寄生虫诱导的 PKB/Akt 和 Bad 磷酸化。此外，用这些 PI3K 抑制剂预处理的 THP-1 细胞显示 Bad 的磷酸化呈剂量依赖性降低，随后无法抑制 Bax 易位，这些细胞也无法克服刚地弓形虫对宿主细胞凋亡的抑制。这些数据表明，PI3K-PKB/Akt 途径可能是刚地弓形虫防止宿主细胞凋亡的主要途径之一，刚地弓形虫磷酸化促凋亡 Bad 蛋白以防止凋亡。
