Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes
Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes
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DOI：
10.1152/ajpgi.1998.275.4.g749
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发表时间：
1998-10-01
影响因子：
4.5
通讯作者：
LaRusso, NF
中科院分区：
文献类型：
--
作者：
Celli, A;Que, FG;LaRusso, NF
胆管细胞是一组称为胆管病的肝脏疾病的目标，包括以导管周围炎症和胆管细胞凋亡为特征的病症。由于炎症与氧化应激有关，我们提出了这样的假设：暴露于氧化应激的胆管细胞将耗尽内源性细胞保护分子，导致胆管细胞凋亡。为了开始验证这一假设，我们探讨了谷胱甘肽（GSH）耗竭、Bcl-2（一种抑制凋亡的原癌基因）表达和非恶性人胆管细胞系凋亡之间的关系。单层人胆管上皮细胞，来自正常肝脏和永生化的SV 40转化，耗尽GSH使用丁硫氨酸亚砜（BSO）。通过定量免疫印迹分析评估Bcl-2表达，并使用DNA结合染料4 '，6'-二脒基-2-苯基吲哚通过荧光显微镜定量细胞凋亡。通过RNA酶保护试验评估Bcl-2信息，并通过脉冲追踪分析评估Bcl-2蛋白合成和半衰期。培养的人胆管细胞暴露于BSO使GSH水平降低93 +/- 3%（P < 0.01）。此外，用BSO处理胆管细胞使Bcl-2水平降低了87 +/- 2%。（P < 0.01），并与凋亡细胞数的时间依赖性增加有关;与未处理的胆管细胞1.5 +/-0.1%相比，培养72小时的细胞凋亡率为11 +/-1%（P < 0.01）。通过在BSO存在下添加谷胱甘肽乙酯来维持GSH水平阻断了BSO处理的胆管细胞中BSO相关的凋亡增加，并且还防止了Bcl-2蛋白的减少。BSO处理胆管细胞没有改变bcl-2 mRNA或Bcl-2蛋白合成的稳态水平。然而，BSO处理的细胞与未处理的细胞相比，Bcl-2蛋白半衰期降低了57%。我们的研究结果表明，使用人胆管细胞系的抗氧化剂，如GSH的细胞水平的降低，导致Bcl-2蛋白的降解增加和细胞凋亡的增加。这些数据提供了氧化应激和胆管细胞凋亡的后果之间的机制联系，一个观察，可能是重要的炎症性胆管病的发病机制。
Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl-2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4',6'-diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 +/- 3% (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 +/- 2% (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; similar to 11 +/- 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 +/- 0.1% in untreated cholangiocytes (P < 0.01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.