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  • Autophagy is a dynamic process that primarily involves

    2020-11-19

    Autophagy is a dynamic process that primarily involves autophagosome formatting and lysosomal degradation, and these processes are controlled by autophagy-related genes (Atg) (Klionsky et al., 2012). The related protein SQSTM1/p62 (sequestosome 1) importantly serves as a signalling director in several signal transduction pathways of autophagy and regulates amounts in signal transduction pathways (Komatsu et al., 2012, Linares et al., 2013). Moreover, this protein is also a selective autophagy receptor for ubiquitinated cargo, including ubiquitinated microbes, damaged mitochondria, and ubiquitinated atrial natriuretic peptide receptor (Bjrky et al., 2005, Rogov et al., 2014). SQSTM1/p62 is usually regarded as an important indicator of autophagy. Previous studies discovered that ER stress activation induces autophagy (Yorimitsu et al., 2006, Fouillet et al., 2012). The present study found that Cr(VI) can induce autophagy in DF-1 cells. This work provides evidence that ER stress is required for Cr(VI)-induced autophagy in DF-1 cells. Moreover, Cr(VI)-induced COX-2 overexpression is attenuated by GRP78 inhibition. This effect suggests that COX-2-mediated autophagy induced by Cr(VI) requires ER stress in the cells. COX-2 inhibition by NS-398 counteracts Cr(VI)-induced autophagy in DF-1 cells. This finding indicates that COX-2 plays an important role in ER-stress-induced autophagy. It has been clear that ER stress can lead to the induction of autophagy, and autophagy initiates a multistep catabolic process to degrade damaged organelles and large protein aggregates (Murrow and Debnath, 2013). Autophagy can be pro-survival and pro-death in cells, and the seemingly conflicting roles actually depending on the interactions between external stimuli and interior adjustments to maintain survival (Cheng and Yang, 2011). Microtubule-associated protein 1 light chain 3 (LC3) exists in two different forms. The free form of LC3 type I (LC3-I) is cytoplasmic, whereas the phosphatidylethanolamine-conjugated form of LC3 type II (LC3-II) is located on autophagosomal membranes. During autophagy, LC3-I is converted to LC3-II, and this process is critical for autophagosome formation (Tanida and Ueno, 2004). In general, LC3-II numbers substantially increase when autophagy occurs, and such an increase is the target of the gold-standard detection of autophagosome formation. LC3-II-related assays for increased LC3-II levels and its co-localization properties with autophagosomal membranes are usually used atrial natriuretic peptide receptor for detecting autophagy. In our present study, the immunoblot analysis results of LC3-II directly reflect the progression of autophagy in Cr(VI)-exposed DF-1 cells. Cr(VI) exposure elevated the LC3-II protein levels in DF-1 cells (Fig. 3). To further investigate the effect of Cr(VI) on autophagic status, we monitored the total p62 amount in DF-1 cells. p62 is a multifunctional protein that binds to LC3, and an enhanced p62 protein level has been regarded as an indicator of autophagy (Jiang and Mizushima, 2015). Thus, p62 protein level changes signal autophagy occurrence from autophagosome accumulation. Cr(VI) intake enhanced the p62 level, and this result indicated the possibility of enhancing autophagic activity. Previous study have shown that Cr(VI) can lead to autophagy in BEAS-2B-Cr cells (Huang et al., 2017) and ER stress in L-02 hepatocytes (Zhang et al., 2017). In this experiment, it has provided evidences that Cr(VI) causes ER stress and autophagy in DF-1 cells, and also found that Cr(VI)-induced autophagy is associated with COX-2 overexpression in DF-1 cells. Further investigation demonstrated that several COX-2 inhibitors, such as NS-398, can reduce the mortality of cells from Cr(VI)-induced injury and autophagy. Additionally, ER stress, which was proven to be required for COX-2 overexpression in cultured cells, occurs with Cr(VI) treatment. Recent studies have suggested that excessive autophagy acts as a destructive mechanism to cell survival (Liu et al., 2016). Herein, the present experiments offer first insight into the cellular mechanisms of Cr-induced autophagy in DF-1 cells. Our study also demonstrates that the eIF2α-ATF4 pathway activates COX-2 transcription to regulate Cr(VI)-induced autophagy.