Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model

Background Activating transcription factor 6 (ATF6) and receptor-interacting protein 3 (RIP3) are important signaling proteins in endoplasmic reticulum (ER) stress and necroptosis, respectively. However, their regulatory relationship and clinical significance are unknown. We investigate the impact of ATF6 on RIP3 expression, and its role in hepatocyte necroptosis in an acute liver injury model. Methods In vivo and in vitro experiments were carried out. LO2 cells were treated with thapsigargin (TG). In vivo, male BALB/c mice were treated with carbon tetrachloride (CCl4, 1 mL/kg) or tunicamycin (TM, 2 mg/kg). Then, the impact of ATF6 or RIP3 silencing on liver injury, hepatocyte necroptosis, and ER stress-related protein expression was examined. Results TG induced ER stress and necroptosis and ATF6 and RIP3 expression in LO2 cells. The knockdown of ATF6 significantly decreased RIP3 expression (p < 0.05) and increased ER stress and necroptosis. The downregulation of RIP3 significantly reduced necroptosis and ER stress (p < 0.05). Similar results were observed in CCl4 or the TM-induced mouse model. The knockdown of ATF6 significantly decreased CCl4-induced RIP3 expression and increased liver injury, necroptosis, and ER stress in mice livers (p < 0.05). In contrast, the downregulation of RIP3 significantly reduced liver injury, hepatocyte necroptosis, and ER stress. Conclusions Hepatocyte ATF6 has multiple roles in acute liver injury. It reduces hepatocyte necroptosis via negative feedback regulation of ER stress. In addition, ATF6 can upregulate the expression of RIP3, which is not helpful to the recovery process. However, downregulating RIP3 reduces hepatocyte necroptosis by promoting the alleviation of ER stress. The findings suggest that RIP3 could be a plausible target for the treatment of liver injury.


Introduction
Hepatocyte necroptosis is considered to be a crucial pathologic pathway involved in several diseases [1]. Hepatocyte necroptosis has been associated with the development of pathologic liver conditions [2,3]. Necroptosis is a caspase-independent mode of cell death that is regulated via multiple proteins that include receptor-interacting protein kinase-1 (RIP1), receptor-interacting protein kinase-3 (RIP3), and mixed lineage kinase domain-like protein (MLKL) [4]. RIP1 and RIP3 bind together forming a RIP1-RIP3 complex. Then, this complex recruits other molecules including the tumor necrosis factor receptor type 1-associated death domain (TRADD), Fas, and tumor necrosis factor receptor-1 (TNFRI) and activates MLKL. MLKL is the terminal mediator for necroptosis [5].
The necroptotic complex that is formed by RIP1, RIP3, and MLKL is crucial for necroptosis [6]. RIP3 binds to the MLKL C-terminus, which stimulates the phosphorylation of the Thr357/Ser358 sites and promotes the activation of the necroptotic complex [7]. Subsequently, activated MLKL migrates to the plasma membrane where it binds to phospholipid-dylinositol lipids via its N-terminal region, causing ion influx through the ion channels and leading to the swelling of organelles, and cell death [8]. Downregulation or silencing MLKL inhibits necroptosis [9]. Therefore, compounds that downregulate MLKL or inhibit its phosphorylation could be promising therapeutic agents. For instance, Necrostatin-1, a RIP1 inhibitor, inhibits necroptosis [10].
Endoplasmic reticulum (ER) stress regulates necroptosis, which is involved in several pathologic conditions [11,12]. ER is important for protein synthesis, folding, and secretion in eukaryotic cells. Cells have evolved a highly regulated mechanism to maintain homeostasis, which helps during folding and the modification of proteins in ER. With the accumulation of misfolded proteins, eukaryotic cells enhance their proteinfolding ability, arrest protein translation, and accelerate protein degradation. With the failure to regain intracellular homeostasis, the cells will activate C/ERB homologous transcription factor protein (CHOP), c-Jun N-terminal kinase (JNK), and/or caspase-12 signaling, which initiates a damaging response [13,14]. In combination, these responses are referred to as an unfolded protein response (UPR) [15]. The UPR is mediated by the inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and protein kinase R-like endoplasmic reticulum kinase (PERK) [16]. Under physiological conditions, glucose-regulated protein 78 (GRP78) binds to IRE1, PERK, and ATF6, and it inhibits their activation. During ER stress, unfolded or misfolded proteins increase and compete with GRP78. Following dissociation from GRP78, IRE1, PERK, and ATF6 are activated, respectively [17]. ATF6 (P90ATF6) dissociates from GRP78 and transfers to the Golgi apparatus, where it is hydrolyzed into an active fragment (P50ATF6).

Induction of ER Stress
In Vitro. LO2 cells were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI-1640 with 10% fetal bovine serum and 1% penicillin/streptomycin. LO2 cells are adherent immortalized human normal hepatocytes with typical morphological characteristics of hepatocytes. They are widely used in experimental studies into liver diseases. To activate ER stress, thapsigargin (TG) was used to treat LO2 cells. TG is an ER membrane Ca 2+ -ATPase that disturbs calcium homeostasis in ER and reduces protein-folding capacity that ultimately induces ER stress in vitro [31,32]. LO2 cells were incubated with dimethyl sulfoxide (DMSO; control group) or 0.5 μmol/L TG (Sigma-Aldrich, USA) for 12, 24, 48, or 72 h. In addition, 1:0 × 10 6 LO2 cells were seeded in a 6well plate, and the target or control short hairpin RNA (shRNA; Syngen Tech Co., Ltd., Beijing, China) were introduced via lentiviral vectors for 48 h (Table 1) and then cells were incubated with TG for 24 h to induce ER stress.

2.2.
Generation of an Acute Liver Injury Mouse Model. Male BALB/c mice (25 ± 3 g; Animal Center of Zunyi Medical University, Guizhou, China) were maintained under specific pathogen-free conditions (12 h light/dark cycle with food and water available ad libitum). A total of 240 animals were  [33]. Following 1 week of acclimatization, mice were randomly assigned to model groups (CCl 4 or TM) and solvent control groups (olive oil for CCl 4 or phosphate-buffered saline (PBS) as the TM solvent control). All treatments were administered via the intraperitoneal route, and experimental outcomes were detected at 12, 24, and 48 h postinjection as detailed previously [30,34]. In the CCl 4 group, mice received 1 mL/kg CCl 4 , and mice in the olive oil control group received 4 mL/kg olive oil. In the TM group, mice received 2 mg/kg TM, and the control group received 5 mL/kg PBS. Alanine aminotransferase (ALT), total bilirubin (TBil), and the area of necrotic liver tissue were used to assess liver injury.
To examine the impact of AFT6 or RIP3 on ER stressmediated apoptosis, mice were administered recombinant adenovirus-associated vector serotype 8 (rAAV8; 2 × 10 10 viral genome copies in 100 μL PBS) that expressed Atf6 or Rip3 short hairpin RNA or control shRNA (Syngen Tech Co., Ltd., Beijing, China; Table 1) via the tail vein as described previously [35]. In the target shRNA or control shRNA groups, CCl 4 was administered 6 weeks after rAAV8 transduction (n = 12). At the end of each experiment, mice were sacrificed by CO 2 euthanasia, and tissue and blood samples were obtained [36].

Cell Viability
Assay. The Cell Titer 96 Aqueous One Solution Cell Proliferation assay kit (Cat. No. 40203ES60; Yeasen; Shanghai Yi San Biotechnology Co., Ltd., Shanghai, China) was used to detect relative LO2 cell viability. The cell suspension (100 μL/well containing approximately 5,000 LO2 cells) was seeded into a 96-well plate (5 replicates per sample). The cell culture plates were placed in the incubator for preculture (37°C, 5% CO 2 ). When 60%-90% cell density was reached, the LO2 cells were subjected to different experimental conditions. At the end of each experiment, the cell culture media was aspirated and 100 μL of the diluted CCK8 reagent was added (diluted with serum-free cell culture solution at a ratio of 1 : 9). The 96-well plate was incubated for 1 h, and the absorbance was recorded at 450 nm on a microplate reader (Bio-Rad, CA, United States). To determine cell viability, we used the following: 2.5. Pathological Analysis of Liver Tissue. Fresh liver tissue (5 mm × 5 mm) was fixed in 4% paraformaldehyde for ≥24 h and dehydrated in a gradient alcohol series, embedded in paraffin, and cut into 5 μm thick liver sections. The paraffin sections were dewaxed and rehydrated and stained with hematoxylin and eosin for the nucleus and cytoplasm, respectively. Sections were then dehydrated and mounted on slides; then, the slides were scanned on a sliced panoramic scanner (Pannoramic DESK/MIDI/250/1000, 3DHIS-TECH, Hungary), observed, and photographed using CaseViewer 2.4 software (3DHISTECH, Hungary). Finally, the area of liver necrosis was analyzed by Image-Pro Plus 6.0 (Media Cybernetics, USA) [30].

Assessment of Liver Function. Serum levels of ALT and
TBil were detected using the rate and diazonium (Beckman Coulter autoanalyzer, AU5800, USA) methods, as detailed previously [38].

Statistical
Analysis. Data were represented as means ± standard deviation. Differences between the different experimental groups were estimated using one-way analysis of variance with Tukey's post hoc analysis (ANOVA), and the least significant difference (LSD) test was used for pairwise comparison. A p value of <0.05 was regarded statistically significant.

Induction of Acute Liver Injury in Mice.
Acute liver injury was induced in mice by CCl 4 or TM injection. Compared with the corresponding control solvent group, CCl 4 and TM significantly increased serum ALT (p < 0:01; Figure 4(a)), TBil levels (p < 0:01; Figure 4(b)), and the necrotic liver area (p < 0:01; Figure 4(c)) at 12, 24, and 48 h after induction, which suggested the successful induction of acute liver injury. Therefore, the expression of ATF6, CHOP, RIP3, and p-MLKL proteins were significantly increased in the liver, which indicated the induction of necroptosis and ER stress along with acute liver injury (p < 0:01; Figure 4(d)).

Discussion
The impact of silencing ATF6 and RIP3 on hepatocyte necroptosis in a hepatocyte model of ER stress and a mouse model of acute liver injury was examined. The results demonstrated that the incubation of LO2 cells with TG induced ER stress and necroptosis and upregulated RIP3 expression. ATF6 downregulation aggravated hepatocyte necroptosis and ER stress and reduced RIP3 expression in TG-induced LO2 cells. On the other hand, the downregulation of RIP3 reduced TG-induced hepatocyte necrosis and p-MLKL and CHOP expression. Comparable results were observed in vivo; ATF6 and RIP3 expression was upregulated along with hepatocyte necroptosis following CCl 4 or TM induction. Similarly, Atf6 downregulation aggravated liver injury, hepatocyte necroptosis, and ER stress and reduced RIP3 expression in CCl 4 -induced mice. However, Rip3 downregulation mitigated liver injury, hepatocyte necroptosis, and ER stress. Taken together, the results imply that ER stress could mediate hepatocyte necroptosis in acute liver injury. Upregulated ATF6 alleviated hepatocyte necroptosis and increased RIP3 expression during ER stress. However, ATF6 and RIP3 differentially control necroptosis in acute liver injury. The downregulation of RIP3 partially increased the protective effects of ATF6 on liver injury. Therefore, targeting RIP3 could be a potential treatment strategy for liver injury.
Controlling hepatocyte necroptosis to treat liver diseases is receiving increased attention [39]. ER stress-mediated necroptosis is independent of the TNFR1 pathway [40]. However, the crosstalk between ER stress and necroptosis and its significance remain unknown. The efficacy of the experimental models, for example, using CCl 4 and TM to induce acute liver injury in mice and the TG-induced ER stress in LO2 cells, was validated in previous reports, and it was previously verified that ER stress mediates necroptosis [30,[41][42][43]. CCl 4 is a well-documented hepatotoxin that induces acute liver injury by oxidative damage via its free radical metabolites [44][45][46]. TM can impede the glycosylation modification of newly synthesized proteins; therefore, it can cause damage to the ER function and induce ER stress in vivo and in vitro [47,48]. In agreement, CCl 4 induced ER 14 BioMed Research International stress and upregulated the expression of p-MLKL. Similarly, the ER stress inducers TM and TG induced p-MLKL expression in vivo and in vitro, respectively. ATF6 is critical for the adaptive response of ER stress, and it is associated with the pathogenesis of various liver conditions [49]. For instance, the loss of ATF6 exacerbates liver steatosis that is caused by acute stress [50]. ATF6 overexpression improves insulin signal transduction and metabolic balance and slows down fatty liver degeneration in obese mice [51]. In addition, ATF6 knock-out mice challenged with TM exhibited sustained CHOP expression and increased liver steatosis [52][53][54]. Following cardiac ischemia-reperfusion injury, the knock-in of ATF6 reduced necrosis and apoptosis and improved cardiac function [55]. In our study, ATF6 knockdown increased the TG-induced p-MLKL expression and aggravated ER stress in LO2 cells. However, knockdown of ATF6 in CCl 4 -induced mice increased the phosphorylation of MLKL, aggravated ER stress, and aggravated liver injury. In combination, this suggested that ATF6 reduces necroptosis in hepatocytes by mitigating ER stress during acute liver injury. In addition, previous studies have reported that ATF6 ameliorates ER stress-mediated liver injury by upregulating sestrin 2 [56].
RIP3, which is a downstream kinase of RIP1, regulates the necroptosis signaling pathway. Phosphorylation of RIP3 at the Ser227 site is the key to its activation. This promotes the recruitment and activation of MLKL, leading to MLKL phosphorylation at Thr357 and Ser358, and therefore, initiating necroptosis signaling [57]. In addition, RIP3 directly phosphorylates RIP1 that further promotes necroptosis [58]. In ischemia-reperfusion injury, RIP3 was the downstream signal for ER stress in myocardial cells and its upregulation eventually leads to necroptosis [59]. The upregulation of RIP3 in chronic alcoholic liver injury leads to necroptosis and steatosis of hepatocytes. In contrast, the downregulation of RIP3 reduces liver injury and liver steatosis [60]. In patients with primary cholestasis, the expression of RIP3 and MLKL is upregulated. Further, RIP3 and MLKL expression is positively correlated with liver injury in mice with bile duct ligation. Knock-out of Rip3 improves necroptosis associated with cholestasis in mice [61]. In this study, the knockdown of Atf6 downregulated the expression of RIP3. Of interest, the knockdown of Rip3 decreased p-MLKL expression and ER stress in CCl 4 -treated mice, which suggested that RIP3 and ATF6 might have different regulatory effects on necroptosis of hepatocytes in acute liver injury. In agreement with previous reports, RIP3 knockdown attenuated liver injury [62]. In addition, previous studies reported that RIP3 regulates ER stress and that knocking down RIP3 has a protective effect on cell damage [63,64]. There have been similar reports in acute myocardial infarction. Here, ZYZ-803, which can release H 2 S and NO slowly, reduced ER stress-related necroptosis by downregulating the RIP3-CaMKII (Ca 2+ -calmodulin-dependent protein kinase) signaling, instead of the classical "RIP1-RIP3-MLKL" axis [65].
Based on results from our research, in the liver, the targeted knockdown of ATF6 reduced RIP3 and aggravated ER stress, and the knockdown of RIP3 reduced ER stress. The effect of RIP3 on ER stress in liver injury appears to be contradictory. However, our results suggested the following: (1) the activation of ATF6 is beneficial in reducing ER stress in acute liver injury; (2) hepatocyte ATF6 upregulated the expression of RIP3; and (3) knockdown of hepatocyte RIP3 promoted the relief of ER stress. However, ATF6 knockdown leads to a decrease in RIP3 expression and aggravates ER stress. ATF6 could have multiple roles in acute liver injury and could have a crucial role in protecting the liver. It reduces hepatocyte necroptosis through the negative feedback regulation of ER stress. In addition, ATF6 upregulates RIP3, which does not help the recovery process. Therefore, the knockdown of RIP3 reduced hepatocyte necroptosis and ER stress (Figure 7). In addition, previous research has shown that ATF6 has a protective and pathological role in certain liver disease models. For example, the loss of ATF6 can prevent steatosis that is caused by chronic ER stress; however, it can enhance steatosis that is caused by acute ER stress [66]. Other researchers observed that ATF6 plays a pathological role, ATF6 prevents alcohol-induced liver steatosis, and ATF6 overexpression induces steatosis in an SREBP-independent manner in a zebrafish alcoholic liver disease model [67]. In combination, AFT6 could have a protective and pathological role in liver disease, which might be related to its role as an important transcription factor by upregulating a variety of downstream target molecules that have different functions.

Conclusion
ATF6 has multiple roles in acute liver injury. It reduces hepatocyte necroptosis in acute liver injury, which could be attributed to the reduction in ER stress. However, ATF6 upregulates the expression of RIP3, which is not beneficial to the recovery from liver injury. The knockdown of RIP3 reduces hepatocyte necroptosis by mitigating ER stress. Therefore, targeting RIP3 could be promising in the future.

RIP3 shRNA RIP3
ATF6 ER stress Hepatocyte necroptosis Liver injury Figure 7: RIP3 shRNA reduces hepatocyte necroptosis. ATF6 plays multiple roles in acute liver injury and plays a dominant role in protecting the liver. It reduces hepatocyte necroptosis through a negative feedback regulation of ER stress. It also upregulates RIP3, which is not favorable to the recovery process. On the other hand, downregulating RIP3 reduces hepatocyte necroptosis by promoting the alleviation of ER stress.