Biomolecules & Therapeutics 2024; 32(3): 341-348  https://doi.org/10.4062/biomolther.2023.174
Endoplasmic Reticulum Stress Activates Hepatic Macrophages through PERK-hnRNPA1 Signaling
Ari Kwon1,2, Yun Seok Kim3, Jiyoon Kim2,* and Ja Hyun Koo1,*
1College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826,
2Department of Pharmacology, Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591,
3Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
*E-mail: jykim@catholic.ac.kr (Kim J), jhkoo@snu.ac.kr (Koo JH)
Tel: +82-2-3147-8358 (Kim J), +82-2-880-7839 (Koo JH)
Received: October 4, 2023; Revised: October 13, 2023; Accepted: October 19, 2023; Published online: April 9, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Endoplasmic reticulum (ER) stress plays a crucial role in liver diseases, affecting various types of hepatic cells. While studies have focused on the link between ER stress and hepatocytes as well as hepatic stellate cells (HSCs), the precise involvement of hepatic macrophages in ER stress-induced liver injury remains poorly understood. Here, we examined the effects of ER stress on hepatic macrophages and their role in liver injury. Acute ER stress led to the accumulation and activation of hepatic macrophages, which preceded hepatocyte apoptosis. Notably, macrophage depletion mitigated liver injury induced by ER stress, underscoring their detrimental role. Mechanistic studies revealed that ER stress stimulates macrophages predominantly via the PERK signaling pathway, regardless of its canonical substrate ATF4. hnRNPA1 has been identified as a crucial mediator of PERK-driven macrophage activation, as the overexpression of hnRNPA1 effectively reduced ER stress and suppressed pro-inflammatory activation. We observed that hnRNPA1 interacts with mRNAs that encode UPR-related proteins, indicating its role in the regulation of ER stress response in macrophages. These findings illuminate the cell type-specific responses to ER stress and the significance of hepatic macrophages in ER stress-induced liver injury. Collectively, the PERK-hnRNPA1 axis has been discovered as a molecular mechanism for macrophage activation, presenting prospective therapeutic targets for inflammatory hepatic diseases such as acute liver injury.
Keywords: PERK, hnRNPA1, Macrophage, ER stress, UPR
INTRODUCTION

Endoplasmic reticulum (ER) stress is a cellular response which play a pivotal role in liver diseases through its distinct downstream signaling cascade called unfolded protein response (UPR). Studies have identified specific UPR pathways to influence the cell-type specific functions in hepatocytes and hepatic stellate cells (HSCs) (Koo and Han, 2021). For example, hepatocyte-specific deletion of IRE1 or ATF6 promotes lipid droplet formation which exacerbates steatohepatitis (Yamamoto et al., 2010; Zhang et al., 2011). Activation of PERK is known to recruit NLRP3 inflammasome to influence hepatocyte survival (Lebeaupin et al., 2015). In HSCs, it has been shown that ER stress increases collagen expression through IRE1-mediated signaling (de Galarreta et al., 2016). On the other hand, inhibition of IRE1 attenuates the TGFβ signaling in HSCs and prevent liver fibrosis development (Heindryckx et al., 2016). Recently, PERK has been also identified as a crucial factor for HSC activation which promotes liver fibrosis (Koo et al., 2016). As such, different UPR factors are responsible for the varying consequences in different cell types within the liver. Therefore, it is crucial to identify the cell type-specific signaling pathways to achieve a comprehensive understanding of complex liver diseases. However, several important gaps in knowledge still exist on the specific roles of UPR in other hepatic cells such as macrophages.

Hepatic macrophages are the main immune component in the liver. Their cardinal functions encompass vigilant responses to pathogenic incursions, orchestration of sterile inflammation, and maintenance of hepatic homeostasis. In the early stages of liver injury, they rapidly expand total hepatic macrophage population by triggering the mobilization of circulating blood-derived monocytes, which differentiate into pro-inflammatory macrophages, amplifying the progression of hepatitis and liver fibrosis (Kazankov et al., 2019). While the substantial role of endoplasmic reticulum (ER) stress in the progression of liver disorders is widely acknowledged, the specific, cell-type-delineated functions of ER stress remain a subject of incomplete elucidation. Nevertheless, studies suggest evidences on the link between ER stress and the functional status of macrophages, albeit with divergent outcomes. For instance, experimentation involving the systemic deletion of CHOP, an apoptosis-inducing UPR effector, has manifested augmented resilience of macrophages against apoptosis which in turn amplifies the inflammation and aggravates steatohepatitis (Malhi et al., 2013). Conversely, investigations employing murine models with myeloid-specific deletion of IRE1 in macrophages have demonstrated a mitigated hepatic ischemia/reperfusion injury, consequent to UPR attenuation (Cai et al., 2022). Correspondingly, administration of an ER stress inhibitor, namely 4-phenyl butyric acid, has been documented to decrease the ratio of M1/M2 macrophage subtypes (Yang et al., 2019). Given the discordant findings, there is a current need to assess whether macrophages assume a protective or deleterious role in hepatic injury induced by ER stress. Furthermore, it is imperative to decipher the underlying molecular mechanisms underpinning these intricate interactions to identify prospective therapeutic targets ameliorating inflammatory hepatic conditions such as acute liver injury and steatohepatitis.

In this study, we aimed to address how ER stress affects hepatic macrophages during liver disease. Our results reveal a sequential response upon acute ER stress which macrophage activation precedes hepatocyte apoptosis. Also, by macrophage depletion methods, the contribution of macrophages in ER stress-induced acute liver injury has been determined to be detrimental. By elucidating PERK-hnRNPA1 as the molecular mechanism underlying ER stress-mediated macrophage activation, our findings provide valuable insights into the field of liver pathology and potentially identify new therapeutic targets.

MATERIALS AND METHODS

Animal experiments

Male C57BL/6 mice at 6 weeks of age were used. To induce ER stress, C57BL/6 mice were killed 12-72 h after an intraperitoneal injection of vehicle or 2 mg/kg tunicamycin (Sigma, MO, USA). Livers were excised and immediately frozen or fixed in 4% formaldehyde for further analysis. For depletion of non-parenchymal cells depletion, mice were intraperitoneally injected with 3 mg/kg gliotoxin (Tocris Bioscience, Bristol, UK) and recovered for 7 days. For specific depletion of macrophages, mice were intravenously injected with 10 mL/kg clodronate liposomes (FormuMax Scientific, CA, USA) and recovered for 7 days. Mice were bred and maintained under specific pathogen-free conditions with a 12-h dark/12-h light cycle and controlled temperature and humidity. Animal studies were approved by the Institutional Animal Care and Use Committee of Seoul National University and were conducted according to guidelines.

Cell culture and treatment

RAW264.7 cells were maintained in DMEM media (Gibco, MA, USA) with 10% FBS (Sigma, #F2442). The cells were cultured at 37°C with in a humidified chamber with 5% CO2.

Delivery of Plasmids and RNAs

Human FLAG-hnRNPA1 clone was purchased from Origene (MD, USA). The constructs encoding for Myc-PERK (#21819, Addgene, MA, USA) and dominant-negative Myc-PERK (#21815, Addgene) were from Dr. David Ron (University of Cambridge, Cambridge, UK). Empty pcDNA3.1 cloning vector was used for mock transfection. For gene silencing, SMARTpool (Dharmacon, CO, USA) and nontargeting siRNA for human PERK, IRE1, ATF6, and ATF4 were used. To modulate miR-18a levels, cells were transfected with miR-18a mimic oligonucleotide (sense: 5’-UAAGGUGCAUCUAGUGCAGAUAG-3’, antisense: 5’-GUAAUCCACGUAGAUCACGUCUA-3’). Scrambled miRNA mimic (sense: 5’-GCAAUUUGGCGUCCUCCACUAA-3’, antisense: 5’-AGUGGAGGACGCCAAAUUCCCU-3’) was used as the transfection control. The cells were transfected with plasmid, small interfering RNA, or miRNA using FuGENE HD (Roche, Basel, Switzerland).

RNA isolation, quantitative PCR

Total RNA was isolated from cells or tissues using TRIzol reagent (Thermo Fisher Scientific, MA, USA) and was reverse-transcribed. Quantitative RT-PCR was performed using a StepOne real-time PCR system (Applied Biosystems, MA, USA) and SYBR Premix Ex Taq II kit (Takara Bio, Kusatsu, Japan). The relative levels of reverse-transcribed mRNAs were normalized based on β-actin levels. After PCR amplification, a melting curve of each amplicon was determined to verify its accuracy.

Western blot

Cells and tissues were lysed in a buffer containing 10 mM Tris-HCl, pH 7.1, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride, supplemented with a protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) as previously described (Koo et al., 2012). The lysates were centrifuged at 10,000 g for 10 min to yield supernatants, which were stored at −70°C until use. Cell or tissue lysates were quantified, separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, IL, USA). For immunoblotting, immobilized proteins of interest were probed with primary antibodies and horse-radish peroxidase-linked secondary antibodies for chemiluminescence detection. Primary antibodies against p-PERK, COX-2, IκBα, PAI-1, and ATF6 were from Santa Cruz Biotechnology (CA, USA). Antibodies for ATF4, CHOP, and Flag were from Cell Signaling Technology (MA, USA). Antibodies for β-actin was from Sigma. Anti-GRP78 antibody was from Abcam (Cambridge, UK).

Bioinformatic Analysis

RIP-ChIP-seq data from a previous report was retrieved and reanalyzed in the study (Papadodima et al., 2013). Gene ontology enrichment and KEGG pathway analyses for the hnRNPA1-binding mRNAs were done by DAVID functional annotation tool. Cytoscape 3.4.0 software (Cytoscape Consortium, https://cytoscape.org/) visualized gene interaction networks of the hnRNPA1-binding mRNAs with protein folding ontology.

Statistical analysis

All statistical analysis was performed using SigmaPlot (SPSS, IL, USA). Criteria for statistical significance were considered to be significant when *p<0.05, and **p<0.01 from Student’s t-test.

RESULTS

ER stress induces hepatic macrophage accumulation and activation

To examine how ER stress impacts macrophages during liver diseases, we employed tunicamycin, a widely used ER stress inducer, to specifically trigger ER stress in mouse liver. A single intraperitoneal injection of tunicamycin induced hepatocyte apoptosis as shown by an increased number of TUNEL-positive cells. The apoptotic hepatocytes peaked at 48 h and gradually decreased by 72 h after injection (Fig. 1A). Intriguingly, F4/80 staining revealed a remarkable rise in hepatic macrophage population at 24 h post-injection, preceding hepatocyte apoptosis. The macrophage population reached its maximum at 48 h, coinciding with the peak of hepatocyte death. This trend was consistent with mRNA expression levels, with F4/80 mRNA increasing from day 1 and peaking on day 2 (Fig. 1B). Similar expression patterns were observed for CD68, another hepatic macrophage marker. An increase in CD11b expression indicated active monocyte recruitment during ER stress-mediated liver injury. These observations suggest that macrophages play a promoting role in ER stress-related liver injury, in addition to the parenchymal cell death potential of ER stress.

Figure 1. ER stress promotes macrophage activation and recruitment in the liver. (A) Immunohistochemistry for F4/80 (top) and TUNEL staining (bottom) in the liver. Relative F4/80 staining intensities are displayed as numbers. C57BL/6 mice were treated with single intraperitoneal injection of tunicamycin (2 mg/kg) and sacrificed after 24, 48, or 72 h. Scale bar, 100 μm. (B) qRT-PCR assay for hepatic macrophage markers. F4/80, CD11b, and CD68 were quantified in the livers from (A) (n=4 each per group). Data represent the mean ± SEM. *p<0.05; **p<0.01, by Student’s t-test. (C) Immunoblottings for macrophage activation markers. RAW264.7 cells were subjected to 2 μg/mL tunicamycin for indicated times.

To further confirm the link between ER stress and macrophages, RAW264.7 macrophage cell line was stimulated with tunicamycin. ER stress induction by tunicamycin caused a time-dependent elevation of COX-2 expression, a marker of macrophage activation. This was preceded by activation of NF-κB signaling, a central pro-inflammatory signaling pathway for COX-2 induction (Yamamoto and Gaynor, 2004), as indicated by IκBα degradation (Fig. 1C). These results support the notion that ER stress can both recruit and further activate macrophages in the liver.

Macrophages contribute to ER stress-induced liver injury

By interacting with other cells in the liver, macrophages play a vital role in either damage or repair mechanisms during the progression of liver disease (Tacke and Zimmermann, 2014). We sought to investigate whether macrophages enhance or prevent ER stress-mediated liver injury. To deplete macrophages in the liver, mice were intravenously administered with clodronate liposomes, which has been extensively used to specifically eliminate macrophages in vivo with high efficiency (Van Rooijen et al., 1996). Surprisingly, the increase in liver-to body weight ratio induced by tunicamycin was completely repressed by macrophage-depletion (Fig. 2A). Elevation of serum ALT and AST was also inhibited in a significant extent (Fig. 2B). On the other hand, clodronate treatment alone was tolerable with no signs of toxicity in both liver weight and serum chemistry. These results indicate that macrophages contribute to and are partly responsible for ER stress-mediated liver damage.

Figure 2. Macrophages contribute to ER stress-induced liver injury and fibrogenesis. (A) Liver-to-body weight ratio. Mice were subjected to intravenous injection of clodronate liposome to deplete macrophage population. After 1 week, the mice were intraperitoneally given 2 mg/kg tunicamycin for 2 days (n=5-7 per group). (B) Blood chemistry. Activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. (C) Immunoblottings for PAI-1 in the liver. Mice were subjected to intraperitoneal injection of gliotoxin to deplete non-parenchymal cell population. After 1 week, the mice were intraperitoneally given 2 mg/kg tunicamycin for 2 days (n=4 per group). Representative immunoblot images (left) and quantification (right) are shown. For A-C, data represent the mean ± SEM. *p<0.05, by Student’s t-test.

Gliotoxin is a potent immunosuppressive mycotoxin which selectively induces cell death in macrophages and other non-parenchymal cells but not in hepatocytes due to their high metabolic capacity (Kweon et al., 2003). To further confirm the results, intraperitoneal injection of gliotoxin was utilized as an independent approach of macrophage depletion. As expected, tunicamycin-induced expression of plasminogen activator inhibitor 1 (PAI-1), a marker for steatohepatitis and fibrosis, was repressed in gliotoxin-treated mice (Fig. 2C). This supported our notion that macrophages are a major contributor of ER stress-induced liver disease progression.

ER stress activates macrophages through PERK

To delineate the molecular basis for ER stress-mediated macrophage activation, three major arms of the UPR signaling pathway were interrogated. Knockdown of PERK, IRE1, ATF6 by their respective siRNA pools showed comparative contribution of each arm. The results showed that ER stress-mediated COX-2 induction in RAW264.7 cells were dependent on PERK, while the roles of IRE1 and ATF6 were minimal (Fig. 3A). Consistently, mRNA expression for IL-1β, a proinflammatory cytokine transcribed by NF-κB, was also dependent on PERK (Fig. 3B). Moreover, transfection with a construct encoding wild-type PERK promoted tunicamycin-induced COX-2 induction, whereas dominant-negative PERK attenuated COX-2 induction (Fig. 3C). This was also confirmed by PERK-dependent mRNA induction for IL-1β and TNFα (Fig. 3D). These results indicate that ER stress activates macrophages through a UPR signaling initiated by ER transmembrane proteins, and probably is controlled by PERK.

Figure 3. PERK-hnRNPA1 degradation signaling is responsible for ER stress-mediated macrophage activation. (A) Immunoblottings for COX-2 after perturbation of unfolded protein response signaling. RAW264.7 cells were transfected with siRNA pools against PERK, IRE1, or ATF6 and treated with 2 μg/mL tunicamycin after 72 h. (B) qRT-PCR assay after PERK silencing. RAW264.7 cells were transfected with siRNA pool against PERK and treated with tunicamycin (2 μg/mL, 16 h) after 72 h. (C) Immunoblottings for COX-2 after modulation of PERK activity. RAW264.7 cells were transfected with wild-type (WT) or kinase dead dominant-negative (DN) Myc-tagged PERK plasmids or empty vector. The cells were treated with tunicamycin (2 μg/mL, 12 h) after 48 h. (D) qRT-PCR assays after modulation of PERK activity. RAW264.7 cells were treated as in (C). (E) Scheme depicting classical PERK signaling pathway. Upon ER stress, PERK is auto-phosphorylated and activated. Then, downstream phosphorylation of eIF2α results in an increased ATF4 translation which in turn promotes transcription of unfolded protein response genes. (F) Immunoblottings for NFκB signaling after ATF4 silencing. RAW264.7 cells were transfected with siRNA pool against ATF4 for 72 h and treated with tunicamycin (2 μg/mL, 12 h). (G) Scheme depicting alternative PERK signaling pathway. Activated PERK can phosphorylate hnRNPA1 as a substrate to accelerate its degradation. This signaling is known to interfere with miR-18a maturation process. (H) Immunoblottings for COX-2 after hnRNPA1 overexpression. RAW264.7 cells were transfected with a vector expressing hnRNPA1 and treated with tunicamycin after 48 h. (I) Immunoblottings for COX-2 after miR-18a mimic oligonucleotide transfection. RAW264.7 cells were transfected with a miRNA mimic for miR-18a then treated with tunicamycin (2 μg/mL, 12 h) after 48 h. For (B) and (D), data represent the mean ± SEM.

PERK-mediated hnRNPA1 degradation is required for macrophage activation

Next, we examined PERK substrates to validate the mechanism of PERK-mediated macrophages activation. The classical perspective of the downstream signaling by PERK includes direct phosphorylation of eiF2α which then induces selective translation of ATF4 transcription factor (Harding et al., 2000). ATF4 then induces UPR target genes to alter cellular functions (Fig. 3E). To confirm whether PERK signals through ATF4 in macrophages, an siRNA was used to silence ATF4. However, ATF4 knockdown failed to reverse tunicamycin-mediated degradation of IκBα (Fig. 3F). The unexpected result led to search for other downstream factors to mediate PERK signaling.

Previous studies have identified hnRNPA1 as another substrate of PERK (Koo et al., 2016; Liao et al., 2021). In hepatic stellate cells, activated PERK directly phosphorylates and destabilizes hnRNPA1, leading to dysregulation of miR-18a maturation (Fig. 3G). To restore hnRNPA1 levels, Flag-tagged wild-type hnRNPA1 was ectopically expressed in cells. This led to nearly complete suppression of COX-2 induction upon tunicamycin treatment in hnRNPA1-transfected cells (Fig. 3H). Importantly, hnRNPA1 expression also attenuated ER stress per se, as indicated by the blocked induction of CHOP, p-PERK, and GRP78. These results indicate that the PERK-hnRNPA1 axis not only regulates macrophage activation in response to ER stress but also facilitates an adaptive UPR response to mitigate ER stress. Meanwhile, transfection with miR-18a showed comparable COX-2 induction, thus ruling out the involvement of miR-18a (Fig. 3I). These findings suggest the participation of another hnRNPA1 substrate in mediating PERK-driven macrophage activation.

hnRNPA1 binds to mRNAs encoding for UPR proteins

hnRNPA1 is an RNA-binding protein that is known to increase mRNA stability and protein translation (Fig. 4A). It binds to the AU-rich element at the 3’-untranslated region which prevent breakdown of its specific target mRNAs (Hamilton et al., 1993). To discover hnRNPA1-binding mRNAs that are relevant to our study, a RIP-chip (RNA-binding protein immunoprecipitation-microarray) profiling data against hnRNPA1 were retrieved from a previous report and reanalyzed (Papadodima et al., 2013). A list of hnRNPA1 included mRNAs of ER protein folding, such as peptidylprolyl isomerase and heat shock proteins (e.g. Ppil3, Dnajc19, Hspa8, and Pdia4) (Fig. 4B). Enriched pathway analysis of the hnRNPA1-binding mRNAs revealed that pathways related to functional maintenance of ER, such as proteasome and ER protein processing pathways (Fig. 4C). Gene ontology analysis also supported this finding; since protein folding, chaperone-mediated protein folding, and protein secretion were among the highly enriched ontologies (Fig. 4D). In particular, genes involved in protein folding were found to be enriched, suggesting that hnRNPA1 may inhibit ER stress by promoting the translation of chaperone proteins, thereby reducing the concentration of misfolded proteins. These genes included the several DNAJC protein family proteins and heat-shock proteins, which have a close functional relevance (Fig. 4E). Taken together, our findings offer a potential mechanism how the PERK-hnRNPA1 axis in macrophages may control ER stress-induced UPR signaling and consequent pro-inflammatory activation.

Figure 4. hnRNPA1 binds to mRNAs encoding UPR-related proteins. (A) Scheme depicting the role of hnRNPA1 in mRNA binding and augmentation of protein translation. (B) List of mRNAs that binds with hnRNPA1. Data was extracted and reanalyzed from a preceding report. (C) KEGG pathway analysis for the hnRNPA1-binding mRNAs. Enriched pathways with p<0.01 are shown. (D) Gene ontology analysis for the hnRNPA1-binding mRNAs. Enriched ontologies with p<0.01 are shown. (E) Interaction map of the mRNAs with gene ontology of “protein folding”. Interaction evidences for individual proteins from each mRNAs are retrieved from String-DB (https://string-db.org/).
DISCUSSION

ER stress is a major driver of liver disease progression (Koo and Han, 2021). While hepatocytes and hepatic stellate cells have been the most studied cell types to study on the role of ER stress in the liver, accumulating evidence directs macrophages as emerging targets of ER stress to induce inflammation and exacerbate parenchymal damage. Our results suggest that ER stress recruits and activates hepatic macrophages, and that this contributes to tunicamycin-induced liver injury. As a molecular mechanism, while PERK was the responsible ER kinase to activate macrophages, a non-canonical downstream signaling through hnRNPA1 was crucial. These suggest an unprecedented role of PERK-hnRNPA1 signaling in the activation of hepatic macrophages, suggesting a novel therapeutic target for liver diseases (Fig. 5).

Figure 5. Schematic diagram illustrating the proposed mechanism from this study.

Studies have shown that ER stress has a potential to influence function and cellular fate of macrophages in different tissues. For example, ER stress induces foam cell differentiation and pro-inflammatory cytokine production in circulating macrophages during atherosclerosis (Yao et al., 2014). In adipose tissue, ER stress has been shown to increase macrophage population and activity as measured by production of IL-6 and TNFα (Kawasaki et al., 2012). In the liver, macrophages are the major component of innate immune cells which secrete pro-inflammatory cytokines in response to acute liver injury. Since most etiologies of both acute and chronic liver diseases commonly accompany ER stress and inflammation (Koo and Han, 2021), one can presume that the role of ER stress in macrophage accumulation and activation at least by some extent. By utilizing two different approaches to deplete macrophage population in our study confirmed that macrophage-induced ER stress in macrophages exerts a critical contribution to amplify liver damage. Our findings suggest that ER stress in macrophages is a crucial factor in the advancement of liver disease.

We also identified the molecular mechanism behind macrophage activation by ER stress. The UPR signaling is activated by the three ER transmembrane sensor proteins: PERK, ATF6, and IRE1. The current view of PERK signaling converges on the selective enhancement of ATF4 translation through eIF2α phosphorylation (Pakos-Zebrucka et al., 2016; Zhang et al., 2022). Nevertheless, while PERK was primarily responsible in ER stress-mediated macrophage activation, ATF4 was dispensable. Instead, hnRNPA1 was found to be the PERK substrate to mediate the regulation of macrophage activity. hnRNPA1 is an RNA-binding protein involved in the regulation of RNA splicing and protein translation, thereby regulating mRNA expression and protein translation (Roy et al., 2017). Our analysis revealed that hnRNPA1 exhibits a preference for mRNAs encoding proteins that support ER function, such as protein folding. Therefore, hnRNPA1 is presumed to be a negative feedback regulator that may enhance ER capacity as a cellular adaptive response. Indeed, in our study hnRNPA1 overexpression in macrophages alleviated ER stress while repressing COX-2 induction. The injured liver contains a mixed population of F4/80+ macrophages, including resident macrophages (namely Kupffer cells) and infiltrating macrophages derived from the bone marrow. The proinflammatory function of NF-κB signaling discussed in this study is similar regardless of macrophage origin. Nonetheless, caution should be taken not to overinterpret the biological implications of the PERK-hnRNPA1 axis as the contribution of the signaling pathway to specific subtypes still requires further verification.

The PERK-hnRNPA1 signaling axis has been investigated as a potential target for inhibiting liver fibrosis (Liao et al., 2021). Our study reveals that this pathway functions not only in HSCs, but also in hepatic macrophages. Chronic liver disease is characterized by a sequential progression from hepatitis to liver fibrosis. In patients, the concurrent development of inflammation and fibrotic lesions are often observed as a result from the coordinated actions of macrophages and HSCs (Friedman et al., 2018). This suggests that targeting a single type of non-parenchymal cells might not suffice. Indeed, most of the therapies against hepatitis and liver fibrosis have failed to prove its efficacy. Therefore, our proposed utilization of the PERK-hnRNPA1 axis that acts in multiple non-parenchymal cells in the liver offers new therapeutic opportunities for liver disease.

Taken together, our findings highlight the role of hepatic macrophages in ER stress-induced liver injury and the cell type-specific function of ER stress in macrophages. Through the identification of the PERK-hnRNPA1 axis as the underlying molecular basis, the results offer new potential drug targets that could prevent hepatocyte death and ameliorate acute liver injury.

ACKNOWLEDGMENTS

This work was supported by National Research Foundation of Korea grants funded by the Korea government (MSIT) (2021R1C1C1013323, 2021R1A4A5033289) as well as by the Creative-Pioneering Researchers Program from Seoul National University.

AUTHOR CONTRIBUTIONS

A.K. and J.H.K. conceived the project and designed the research, performed the studies, analyzed the data, and wrote the manuscript. Y.S.K. developed methodology and reviewed the manuscript. J.K. and J.H.K. provided administrative support and obtained funding. J.H.K. supervised overall data.

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