TXNIP mediates high glucose-induced mitophagic flux and lysosome enlargement in human retinal pigment epithelial cells
TXNIP mediates high glucose-induced mitophagic flux and lysosome enlargement in human retinal pigment epithelial cells
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DOI：
10.1242/bio.038521
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发表时间：
2019-04-01
期刊：
影响因子：
2.4
通讯作者：
Singh, Lalit P.
中科院分区：
文献类型：
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作者：
Devi, Takhellambam S.;Yumnamcha, Thangal;Singh, Lalit P.
硫氧还蛋白相互作用蛋白（TXNIP）在氧化应激、炎症、细胞凋亡和糖尿病视网膜病变（DR）的发病机制中起着重要作用。然而，TXNIP在高糖诱导的视网膜色素上皮（RPE）功能障碍中的作用仍不清楚。在这里，我们表明，当与人RPE细胞系（ARPE-19）和原代人RPE（HRPE）细胞中的低葡萄糖（LG; 5.5 mM）相比时，高葡萄糖（HG; 25 mM）在mRNA和蛋白质水平上显著增加TXNIP表达。TXNIP上调与线粒体有关！膜去极化，碎裂和线粒体吞噬流到溶酶体。我们使用表达mt-Keima的RPE细胞的共聚焦活细胞成像来测量线粒体吞噬通量，mt-Keima是一种在线粒体（碱性或中性pH）中发射绿色光和在酸性溶酶体中发射红光的珊瑚蛋白。我们观察到一个拉长的线粒体网络的绿色mt-Keima在LG下，这是破碎的HG。在ARPE-19和HRPE细胞中，红色mt-Keima在LG下作为小的点状聚集体积累在溶酶体中，而在HG下它们显著增大（2 - 3倍）。HG下的溶酶体增大通过ARPE-19和HRPE细胞中的溶酶体膜蛋白LAMP 1-mCherry表达进一步说明。此外，HG导致ARPE-19细胞中溶酶体组织蛋白酶L失活和促炎性caspase-1激活。通过shRNA敲低TXNIP可防止HG下的线粒体碎片化、线粒体吞噬通量和溶酶体扩大。此外，抗氧化剂N-乙酰半胱氨酸（NAC）和Amlexanox（Amlx），蛋白激酶TBK 1和线粒体吞噬衔接子Optineurin（Optn）和Sequestosome 1（p62/SQSTM 1）的抑制剂，可防止线粒体吞噬通量和溶酶体扩大。这些结果表明，TXNIP介导了高糖对RPE的几种有害作用，这可能与DR的发展有关。
Thioredoxin-interacting protein (TXNIP) plays a critical role in oxidative stress, inflammation, apoptosis and the pathogenesis of diabetic retinopathy (DR). However, the role of TXNIP in high glucose-induced retinal pigment epithelium (RPE) dysfunction is still unknown. Here, we show that high glucose (HG; 25 mM,) significantly increases TXNIP expression at both the mRNA and protein levels when compared to low glucose (LG; 5.5 mM) in a human RPE cell line (ARPE-19) and primary human RPE (HRPE) cells. TXNIP upregulation is associated with mitochondria! membrane depolarization, fragmentation and mitophagic flux to lysosomes. We used confocal live-cell imaging of RPE cells expressing mt-Keima, a coral protein that emits green light in mitochondria (alkaline or neutral pH) and red light in the acidic lysosome, to measure mitophagic flux. We observed an elongated mitochondrial network of green mt-Keima under LG, which is fragmented in HG. Red mt-Keima accumulates in lysosomes as small punctate aggregations under LG in both ARPE-19 and HRPE cells, whereas they are significantly enlarged (two- to threefold) under HG. Lysosomal enlargement under HG is further illustrated by lysosomal membrane protein LAMP1-mCherry expression in both ARPE-19 and HRPE cells. Furthermore, HG causes lysosomal cathepsin L inactivation and pro-inflammatory caspase-1 activation in ARPE-19 cells. TXNIP knockdown by shRNA prevents mitochondrial fragmentation, mitophagic flux and lysosome enlargement under HG. In addition, antioxidant N-acetylcysteine (NAC) and Amlexanox (Amlx), an inhibitor of protein kinase TBK1 and of the mitophagic adaptors Optineurin (Optn) and Sequestosome 1 (p62/SQSTM1), prevent mitophagic flux and lysosome enlargement. These results suggest that TXNIP mediates several deleterious effects of high glucose on RPE, which may be implicated in the development of DR.