Platelet-activating factor-induced apoptosis is blocked by Bcl-2 in rat intestinal epithelial cells.
Platelet-activating factor-induced apoptosis is blocked by Bcl-2 in rat intestinal epithelial cells.
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DOI：
10.1152/ajpgi.00182.2003
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发表时间：
2004-02
期刊：
影响因子：
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通讯作者：
Jing Lu;M. Caplan;A. P. Saraf;Dan Li;L. Adler;X. Liu;T. Jilling
中科院分区：
文献类型：
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作者：
Jing Lu;M. Caplan;A. P. Saraf;Dan Li;L. Adler;X. Liu;T. Jilling
血小板活化因子（PAF）是炎症性肠病（IBD）发病机制中的重要介质，但PAF引起粘膜损伤的机制尚不清楚。为了确定细胞凋亡和Bcl-2家族的凋亡调控基因产物是否在PAF诱导的粘膜损伤中发挥作用，我们在乳糖诱导型启动子的控制下，在大鼠小肠上皮细胞-6中稳定和有条件地过表达Bcl-2。Western印迹分析和免疫组织化学用于验证诱导型Bcl-2和分析Bcl-2和Bcl-2家族的促凋亡成员Bax的亚细胞分布。采用酶联免疫吸附法（ELISA）定量DNA片段化，荧光肽底物法测定caspase活性，JC-1法和荧光数字成像法测定线粒体膜电位。Bcl-2的表达是高度诱导乳糖类似物异丙基-β-（d）-硫代半乳糖苷（IPTG），并主要定位于线粒体。在bcl-2不表达的情况下，PAF处理后，Bax易位到线粒体，线粒体膜电位在1h内崩溃，随后caspase-3激活，6 h达到峰值，随后DNA片段化在18 h达到最大值。IPTG诱导bcl-2表达后，PAF未能诱导DNA断裂、caspase-3激活、Bax易位或线粒体膜电位崩溃。这些数据是第一次表明，PAF可以激活肠上皮细胞凋亡机制，通过涉及Bax易位和线粒体膜电位的崩溃，这两个事件是由bcl-2的表达水平控制。更好地了解PAF和Bcl-2家族凋亡调节因子在上皮细胞死亡中的作用可能有助于设计更好的IBD治疗或预防策略。
Plateletactivating factor (PAF) is a key mediator in pathogenesis of inflammatory bowel diseases (IBDs) but mechanisms of PAF-induced mucosal injury are poorly understood. To determine whether apoptosis and the Bcl-2-family of apoptosis regulatory gene products play a role in PAF-induced mucosal injury, we stably and conditionally overexpressed bcl-2 in rat small intestinal epithelial cells-6 under the control of a lactose-inducible promoter. Western blot analysis and immuno-histochemistry were used to verify inducible Bcl-2 and to analyze Bcl-2 and a proapoptotic member of the Bcl-2 family, Bax, subcellular distribution. DNA fragmentation was quantified by ELISA, caspase activity was measured by using fluorogenic peptide substrates, and mitochondrial membrane potential was assayed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and fluorescence digital imaging. Bcl-2 expression was highly inducible by lactose analog isopropyl-beta-(d)-thiogalactoside (IPTG) and was localized predominantly to mitochondria. In the absence of bcl-2 overexpression and after treatment with PAF, Bax translocated to mitochondria, and mitochondrial membrane potential collapsed within 1 h, followed by caspase-3 activation, which peaked at 6 h with an ensuing DNA fragmentation maximizing at 18 h. After IPTG-induction of bcl-2 expression, PAF failed to induce DNA fragmentation, caspase-3 activation, Bax translocation, or a collapse of mitochondrial membrane potential. These data are the first to show that PAF can activate apoptotic machinery in enterocytes via a mechanism involving Bax translocation and collapse of mitochondrial membrane potential and that both of these events are under control by bcl-2 expression levels. A better understanding of the role of PAF and Bcl-2 family of apoptosis regulators in epithelial cell death might aid design of better therapeutic or preventive strategies for IBDs.