Lee Y I, Kang-Park S, Do S I, Lee Y I
Liver Cell Signal Transduction Research Unit, Animal Cell and Medical Glycobiology Research Unit, Bioscience Research Division, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Taejon 305-600, Korea.
J Biol Chem. 2001 May 18;276(20):16969-77. doi: 10.1074/jbc.M011263200. Epub 2001 Mar 1.
The hepatitis B virus-X (HBx) protein is known as a multifunctional protein that not only coactivates transcription of viral and cellular genes but coordinates the balance between proliferation and programmed cell death, by inducing or blocking apoptosis. In this study the role of the HBx protein in activation of phosphatidylinositol 3-kinase (PI3K) was investigated as a possible cause of anti-apoptosis in liver cells. HBx relieved serum deprivation-induced and pro-apoptic stimuli-induced apoptosis in Chang liver (CHL) cells. Treatment with 1-d-3-deoxy-3-fluoro-myo-inositol, an antagonist to PI3K, which blocks the formation of 3'-phosphorylated phosphatidyl inositol in CHL cells transformed by HBx (CHL-X) but not normal Chang liver (CHL) cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI3K, stimulated apoptosis in HBx-transformed CHL cells but not in normal cells, confirming that HBx blocks apoptosis through the PI3K pathway. The serine 47 threonine kinase, Akt, one of the downstream effectors of PI3K-dependent survival signaling was 2-fold higher in HBx-transformed CHL (CHL-X) cells than CHL cells. Phosphorylation of Akt at serine 473 and Bad at serine 136 were induced by HBx, which were specifically blocked by wortmannin and dominant negative mutants of Akt and Bad, respectively. We also demonstrated that HBx inhibits caspase 3 activity and HBx down-regulation of caspase 3 activity was blocked by the PI3K inhibitor. Regions required for PI3K phosphorylation on the HBx protein overlap with the known transactivation domains. HBx blocks apoptosis induced by serum withdrawal in CHL cells in a p53-independent manner. The results indicate that, unlike other DNA tumor viruses that block apoptosis by inactivating p53, the hepatitis B virus achieves protection from apoptotic death through a HBx-PI3K-Akt-Bad pathway and by inactivating caspase 3 activity that is at least partially p53-independent in liver cells. Moreover, these data suggest that modulation of the PI3K activity may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in human hepatocellular carcinoma.
乙肝病毒X蛋白（HBx）是一种多功能蛋白，它不仅能共激活病毒和细胞基因的转录，还能通过诱导或阻断细胞凋亡来协调细胞增殖与程序性细胞死亡之间的平衡。在本研究中，对HBx蛋白在激活磷脂酰肌醇3激酶（PI3K）中的作用进行了研究，这可能是肝细胞抗凋亡的一个原因。HBx可减轻血清剥夺诱导的凋亡以及促凋亡刺激诱导的张氏肝癌（CHL）细胞凋亡。用1-d-3-脱氧-3-氟-肌醇（一种PI3K拮抗剂）处理，它能阻断HBx转化的CHL细胞（CHL-X）中3'-磷酸化磷脂酰肌醇的形成，但对正常张氏肝癌细胞（CHL）无效，结果显示细胞活力显著丧失并有凋亡迹象。同样，用PI3K抑制剂渥曼青霉素处理，可刺激HBx转化的CHL细胞凋亡，但对正常细胞无效，这证实了HBx通过PI3K途径阻断凋亡。丝氨酸47苏氨酸激酶Akt是PI3K依赖性生存信号的下游效应器之一，在HBx转化的CHL（CHL-X）细胞中的水平比CHL细胞高2倍。HBx诱导Akt在丝氨酸473位点以及Bad在丝氨酸136位点的磷酸化，它们分别被渥曼青霉素以及Akt和Bad的显性负突变体特异性阻断。我们还证明HBx抑制半胱天冬酶3的活性，并且PI3K抑制剂可阻断HBx对其活性的下调作用。HBx蛋白上PI3K磷酸化所需区域与已知的反式激活结构域重叠。HBx以不依赖p53的方式阻断CHL细胞中血清剥夺诱导的凋亡。结果表明，与其他通过使p53失活来阻断凋亡的DNA肿瘤病毒不同，乙肝病毒通过HBx-PI3K-Akt-Bad途径以及至少部分在肝细胞中不依赖p53的方式使半胱天冬酶3失活来实现对凋亡死亡的保护。此外，这些数据表明调节PI3K活性可能是对抗人类肝细胞癌中凋亡发生的一种潜在治疗策略。
