Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak
Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak
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DOI：
10.1074/jbc.m112.364281
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发表时间：
2012-07-06
影响因子：
4.8
通讯作者：
Oh, Young J.
中科院分区：
文献类型：
--
作者：
Lim, Junghyun;Lee, Yunsu;Oh, Young J.
BAK是一种典型的促凋亡蛋白，表达于多种组织和细胞中。最近的研究表明，Bcl2家族蛋白调节细胞凋亡和自噬。为了研究Bak是否以及如何在自噬相关事件中发挥调节作用，我们用K+/H+离子载体黑素处理了独立的细胞系，包括MN9D神经元细胞。黑素处理MN9D细胞后，Lc3-II和p62水平升高，同时caspase激活。超微结构检查显示黑素处理的细胞内有自噬空泡和肿胀的空泡积聚。我们进一步发现，在黑素处理的细胞中，LC3-II的积累是自噬通量受损和LC3-II降解中断的结果。在这种细胞死亡模式中，各种形式的Bak的瞬时和稳定过表达都发挥了保护作用，但它并不抑制黑素介导的caspase-3的激活程度。随后的生化和电子显微镜研究表明，在黑素处理的细胞中，过表达的Bak维持了自噬通量，并减少了膨胀的空泡所占的面积。在黑素处理的非神经细胞和另一种质子离子载体诱导的细胞死亡范例中也得到了类似的结果。综上所述，我们的研究表明，Bak在离子载体诱导的细胞死亡中的保护作用可能与其对维持自噬通量和液泡动态平衡的调节作用密切相关。
Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K+/H+ ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.