Tunicamycin was purchased from Tocris (Minneapolis, MN, USA). CCl4 was purchased from Guoyao (Beijing, China). LPS and D-GalN were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against ATF4, p-eIF2α and Bip were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against eIF2α and GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).

Male C57BL/6 mice (10 weeks, 20–22 g) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). The use of animals was approved by the Ethics Committee of Southwest Medical University on Animal Care (Sichuan, China).

Hydrodynamic injection was performed as described in the report of Chen and Calvisi (16). In brief, 10 µg ATF4-targeting CRISPR-Cas9 plasmid, ATF4 overexpression plasmid or empty vector were diluted in 2 ml saline (0.9% NaCl), filtered through a 0.22 µm filter and injected into the lateral tail vein of 10-week-old male C57BL/6 mice in 5 to 7 sec.

Male C57BL/6 mice (6 mice per group) were injected intraperitoneally with CCl4 (4 ml/kg, 5% w/v dissolved in olive oil) three times a week for 2 weeks as previously described (17). The mice were killed 24 h after the final injection of CCl4, and liver tissues were harvested for analysis.

Male C57BL/6 mice (6 mice per group) were injected intraperitoneally with LPS (50 µg/kg) and D-GalN (800 mg/kg, phosphate buffer saline as control) and killed 6 h after LPS/D-GalN injection (18).

Liver tissues of mice were fixed in 4% formalin at room temperature for at least 24 h, embedded in paraffin and cut into 5 µm sections. Liver sections were deparaffinized and stained with hematoxylin and eosin (H&E) for morphologic analysis. Sirius red staining was performed according to the usual method and the positive area was quantified with Image J software.

Total RNA was isolated with TRIzol reagent (Invitrogen; Thermo Fischer Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The reverse transcription reactions were carried out using the M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. sqPCR was performed by running the products on a 1% (for ATF4 and 18S) or 4% (for XBP1) agarose gel. RT-qPCR analyses were performed using SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan) as previously described (17). Results were normalized with 18S and quantified using the 2^−∆∆Cq method (19). The primers used are as follows: Mouse ATF4-forward: 5′-TCCTGAACAGCGAAGTGTTG, andmouse ATF4-reverse: 5′-AGAGCTCATCTGGCATGGTT-3′; mouse XBP1-forward: 5′-TGCTGAGTCCGCAGCAGGTG-3′, and mouse XBP1-reverse: 5′-ACTAGCAGACTCTGGGGAAG-3′; mouse 18S-forward: 5′-CGGCTACCACATCCAAGGAA-3′, and mouse 18S-reverse: 5′-GCTGGAATTACCGCGGCT-3′.

Mouse tissues were lysed in Triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF, 10 mM NaF, 5 mg/ml aprotinin, 20 mM leupeptin, and 1 mM sodium orthovanadate) and centrifuged at 4°C, 12,000 × g for 15 min. Protein concentrations of the supernatant were measured using the BCA assay. Protein samples were denatured with 4× SDS-loading buffer (200 mM Tris, pH 6.8, 8% SDS, 400 mM DTT, 0.4% bromophenol blue, 40% glycerol) at 100°C for 5 min and subjected to standard SDS-PAGE and western blot analysis as previously described (17).

Results are expressed as the mean ± standard deviation. Statistical analysis was performed using Student's t-test and Excel software (version 2010; Microsoft Corporation, Redmond, WA, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the expression of ATF4 in vivo, we evaluated both protein and mRNA levels of ATF4 in mouse tissues, including liver, heart, kidney, lung, stomach, spleen, and small and large intestine. Interestingly, the western blot results showed ATF4 protein is highly expressed in mouse liver, while being almost nondetectable in other tissues (Fig. 1A). However, the RNA levels of ATF4 in these tissues are comparably high (Fig. 1B).

Considering that ATF4 is conventionally regulated by eIF2α, we analyzed the phosphorylation level of eIF2α in mouse tissues by western blotting. Our results showed that phospho-eIF2α levels are very low in the tissues tested (Fig. 2A), which seemed contradictory with the high protein level of ATF4 in the liver. To clarify whether ATF4 can be upregulated by ER stress in the mouse liver, we treated mice with tunicamycin. As shown in Fig. 2B, tunicamycin treatment caused XBP1 mRNA splicing, suggesting the induction of the unfolded protein response. Next, we determined the eIF2α/ATF4 signal in mouse liver and lung upon tunicamycin treatment. The results showed that tunicamycin significantly promoted eIF2α phosphorylation, and increased ATF4 and Bip protein levels in mouse lungs (Fig. 2C). In the liver tissue, phosphorylation of eIF2α and expression of Bip were increased upon tunicamycin administration as expected. However, the ATF4 protein level decreased in a time-dependent manner after tunicamycin treatment (Fig. 2C). These results indicated that the high levels of liver ATF4 protein present in the liver are independent of eIF2α or ER stress.

It was interesting to find that ATF4 protein displayed high levels of expression in the mouse liver and was nonconventionally regulated. We therefore investigated the role of ATF4 in liver injury. Animal models of liver injury are commonly used in research, for example CCl4 is a classical hepatotoxicant, which is used to induce chronic liver injury and liver fibrosis (20). Similarly, LPS plus D-GalN is a well-known acute liver injury model (21). Thus, CCl4 was used to establish chronic liver injury while LPS/D-GalN was used to induce acute liver injury. The western blot assay demonstrated that ATF4 protein was decreased significantly following repeated CCl4 exposure, while the mRNA level of ATF4 was not significantly changed (Fig. 3A and B). In addition, the ATF4 protein decreased markedly after 6 h of LPS/D-GalN treatment (Fig. 3C). In contrast, the mRNA of ATF4 did not change significantly (Fig. 3D). These results suggested that ATF4 protein is downregulated in response to both chronic and acute liver injury.

Next, to investigate effects of ATF4 on liver injury, ATF4 targeting a CRISPR-Cas9 plasmid (ATF4-cri) was constructed and injected through the tail vein to knockdown the expression of ATF4 in the liver. As shown in Fig. 4A and B, the ATF4-cri plasmid efficiently lowered the liver ATF4 expression at both mRNA and protein levels. After injection with ATF4-cri plasmid or control plasmid, mice were challenged with CCl4 or LPS/D-GalN. Serum transaminase analysis revealed that knockdown of ATF4 by ATF4-cri significantly increased CCl4-induced levels of AST and ALT compared with controls (Fig. 4B). Similar results were obtained in the LPS/D-GalN model (Fig. 4C). These data suggested ATF4 inactivation sensitizes mice to CCl4 and LPS/D-GalN induced liver injury, indicating a protective role for ATF4 in the liver.

To reveal the basis for the increased liver injury by ATF4 inactivation, mouse liver sections were subjected to histopathological examination. Hematoxylin and eosin (H&E) staining results revealed more serious hepatocellular necrosis and morphological alterations in the ATF4-cri group after CCl4 treatment (Fig. 5A). In addition, we found enhanced liver fibrosis in the ATF4-cri group mice as evidenced by increased intensity of Sirius red staining (Fig. 5B). H&E staining of LPS-treated liver sections showed markedly more hemorrhage, necrosis and inflammatory cell infiltration in ATF4-cri-treated mice livers (Fig. 5C). These data demonstrated that ATF4 suppression augmented hepatocyte damage and the inflammatory response in both the CCl4 and LPS/D-GalN models. The c-Jun-N-terminal kinase (JNK) is a mitogen-activated protein kinase family member that plays important roles in the regulation of cell death, survival, and inflammation (22). We therefore explored a possible role for JNK in our model. The results showed that both CCl4 and LPS/D-GalN treatment lead to the activation of JNK (Fig. 5D). More importantly, ATF4 suppression increased the activation of JNK induced by CCl4 and LPS/D-GalN (Fig. 5D). These results suggested that ATF4 plays a protective role of in the liver, in part, through regulating JNK signaling.

To verify the protective role of ATF4 in the liver, we investigated the effects of ATF4 overexpression on liver injury induced by CCl4 and LPS/D-GalN. After injection with ATF4 overexpression plasmid (ATF4-ov) or control plasmid, mice were challenged with CCl4 or LPS/D-GalN. Serum transaminase analysis revealed that overexpression of ATF4 significantly decreased CCl4 induced AST and ALT elevation compared with controls (Fig. 6A). Similar results were obtained in the LPS/D-GalN model (Fig. 6B). These data thus further confirm the protective role of ATF4 in the liver.