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  • The UPR is composed of three different pathways

    2020-08-10

    The UPR is composed of three different pathways that fall under the control of three respective ER transmembrane proteins: PERK, IRE1α (inositol-requiring enzyme 1α) and ATF6 (activating transcription factor 6). As a starting signal for the UPR, misfolded proteins induce the release of GRP78 from these ER transmembrane proteins. Free of GRP78, all three ER transmembrane proteins are activated and initiate signaling [25], [26]. The UPR is activated by three main stress sensors, IRE1, ATF6 and PERK, which result in multiple cellular responses and are summarized in Fig. 7 [27]. Active PERK phosphorylates eIF2a and elicits various cellular responses such as protein synthesis inhibition and apoptotic signal activation [29]. While PERK inhibits general protein synthesis through eIF2α phosphorylation, there is still active translation of ATF4, a transcription factor that regulates both pro-survival and pro-apoptotic genes that participate in the UPR [30]. On the one hand, ATF4 forms a complex with the phosphorylated form of CREB1 to bind to the promoter region of the pro-survival gene, GRP78 [31]. On the other hand, the increasing translation of ATF4 induces the expression of downstream gene CHOP, a transcription factor that is critical in supporting the ERS-induced apoptotic program [32], [33]. The transcriptional induction of CHOP is controlled by the PERK-eIF2a-ATF4 axis in a positive manner [34]. To explore the underlying mechanisms of the EWAs in ERS-induced hepatocyte injury, we focused on the intervention of the EWAs in PERK-eIF2a-ATF4 pathway.
    Conclusions In conclusion, tunicamycin-induced ERS model in L02 szl is successfully established. The best condition of tunicamycin treatment is using a 60μg/mL dose for 24h treatment. The EWAs has therapeutic effects in injured hepatocyte through promoting proliferation and inhibiting apoptosis. The mechanism may have a connection with inhibiting the PERK-eIF2a-ATF4 pathway and relieving ERS. These findings suggest the EWAs might be a potential hepatoprotective which is warranted further investigations.
    Conflicts of interest
    Acknowledgements This research was supported by the National Natural Science Foundation of China (No. U1204826) and the Science and technology project of Henan Provincial Health Department (No. 200903110).
    Introduction PKR is a serine/threonine kinase composed of an N-terminal regulatory domain that contains two dsRNA binding motifs (DRBMs) and a C-terminal kinase domain (Meurs et al., 1990, Nanduri et al., 1998). These domains are connected by a spacer that provides an interface for dimerization (McKenna et al., 2007). It has been proposed that in the unphosphorylated state, the N-terminal regulatory domain interacts with the C-terminal catalytic domain to inhibit kinase activity (Nanduri et al., 2000). Activation of PKR by dsRNA results in the formation of dimers that are stabilized by autophosphorylation at multiple residues, including Thr446 and Thr451 that are located within the activation loop of the kinase domain and essential for PKR activation (Romano et al., 1998). To date, 18 PKR phosphorylation sites have been identified. Most are serine residues but some are threonine or tyrosine residues (Su et al., 2006, Toth et al., 2006). Active PKR dimers eject the activating dsRNA, presumably, due to phosphorylation of N-terminal residues and then phosphorylate eIF2a (Jammi and Beal, 2001). PKR is constitutively and ubiquitously expressed at low levels due to a kinase conserved sequence (KCS) site in its promoter (Toth et al., 2006). PKR expression is upregulated by Type I IFN which can be produced in response to a viral infection. The majority of PKR is located in the cytoplasm where a portion is associated with ribosomes. Some of the PKR in the nucleus associates with nucleoli (MacQuillan et al., 2009, Tanaka and Samuel, 1994, Toth et al., 2006). A ternary complex consisting of GTP-eIF2 and a methionyl-tRNA delivers the charged initiator tRNA to the 40S ribosomal subunit of the 43S preinitiation complex but translation initiation requires the hydrolysis of the eIF2-bound GTP to a GDP (Hershey, 1991, Majumdar and Maitra, 2005). Under stress conditions, the alpha-subunit of eIF2 is phosphorylated by one of four eIF2a kinases: general control non-repressed 2 (GCN2), heme-regulated inhibitor (HRI), PKR-like endoplasmic reticulum (ER) kinase (PERK), or PKR (Kaufman, 1999). The eIF2a kinases share a conserved kinase domain that mediates eIF2a phosphorylation, but each responds to a different stress due to its unique regulatory domain (Kaufman, 1999). Phosphorylation of eIF2a on Ser51 leads to the formation of a high-affinity complex with the guanine exchange factor, eIF2B. This inhibits the exchange of GDP with GTP and “stalls” the preinitiation complexes on mRNAs (Sudhakar et al., 2000). Phosphorylation of as little as 20% of eIF2a significantly reduces the synthesis of most cellular proteins (Sudhakar et al., 2000). In virus-infected cells, PKR is activated by viral dsRNA. However, PKR can also be activated by Type I or II IFN by a mechanism mediated by the activated JAKs of the IFN receptor complex (Su et al., 2007), by heparin oligosaccharides, or by IL-3 withdrawal (Toth et al., 2006). PKR activation by peroxide or arsenite treatment is mediated through interaction of the activation domain of PACT with the N-terminal domain of PKR (Ito et al., 1999, Patel et al., 2000).