Inhibition of CLIC4 Enhances Autophagy and Triggers Mitochondrial and ER Stress-Induced Apoptosis in Human Glioma U251 Cells under Starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.

CLIC4/mtCLIC（氯离子细胞内通道蛋白，chloride intracellular channel protein）定位于线粒体、内质网（endoplasmic reticulum, ER）、细胞核与细胞质，并参与应激诱导的细胞凋亡应答。细胞凋亡与自噬作为程序性细胞死亡的两大主要类型，在特定应激条件下存在相互关联。然而，CLIC4在自噬调控中的作用仍有待明确。

本研究证实，U251细胞在饥饿应激下，CLIC4蛋白表达上调并发生核转位。在饥饿条件下，转染CLIC4小干扰RNA（small interfering RNA, siRNA）可通过提升LC3-II蛋白水平与斑点聚集，增强U251细胞的自噬活性。在此状态下，14-3-3ε亚型与CLIC4的相互作用被阻断，进而导致Beclin 1过度激活，最终进一步激活自噬通路。

此外，在饥饿应激下抑制CLIC4的表达，可触发涉及Bax/Bcl-2与细胞色素c释放的线粒体凋亡，同时上调CHOP与半胱天冬酶-4（caspase-4）的表达，诱发内质网应激介导的细胞凋亡。

上述结果表明，CLIC4核转位是细胞饥饿应答的核心组成部分。在饥饿条件下，抑制CLIC4的表达可增强自噬，并促进线粒体与内质网应激诱导的细胞凋亡。