Gα12 overexpression in hepatocytes by ER stress exacerbates acute liver injury via ROCK1-mediated miR-15a and ALOX12 dysregulation

Rationale: Liver injury must be further characterized to identify novel therapeutic approaches. Endoplasmic reticulum (ER) stress may cause hepatocyte death. Gα12 affects cell viability and its expression varies depending on physiological conditions. This study investigated whether hepatocyte-specific Gα12 overexpression affects acute liver injury, and if so, what the underlying mechanisms and treatment strategies are. Methods: All experiments were performed using human liver, hepatocytes, and toxicant injury models with Gna12 KO and/or hepatocyte-specific Gα12 overexpression. RNA-sequencing, immunoblotting, immunohistochemistry, reporter assays, and mutation assays were conducted. Results: Hepatic Gα12 was overexpressed in mice challenged with acetaminophen or other ER stress inducers or in patients with acute liver injury or fibrosis/cirrhosis. Several Gα12 and ER-associated pathways were identified using transcriptomic analysis. Acetaminophen intoxication was characterized by lipid peroxide-induced ferroptosis and was less severe in Gα12-deficient animals and cells. Conversely, Gα12 overexpression in wild-type or Gna12 KO hepatocytes increased hepatotoxicity, promoting lipid peroxidation, inflammation, and ferroptosis. IRE1α-dependent Xbp1 transactivated Gna12. Moreover, Gα12 overexpression enhanced the ability of acetaminophen to induce ALOX12, while downregulating GPX4. The level of miR-15a, herein identified as an ALOX12 inhibitor, was decreased. siRNA knockdown or pharmacological inhibition of ROCK1 prevented dysregulation of ALOX12 and GPX4, rescuing animals from toxicant-induced ferroptosis. These changes or correlations among the targets were confirmed in human liver specimens and datasets of livers exposed to other injurious medications. Conclusions: Gα12 overexpression by ER stress facilitates hepatocyte ferroptosis through ROCK1-mediated dysregulation of ALOX12, and miR-15a, supporting the concept that inhibition of Gα12 overexpression and/or ROCK1 axis may constitute a promising strategy for acute liver injury.


Analysis of human samples
For the analysis of drug-induced liver injury (DILI) samples, human liver samples were obtained from donors and recipients undergoing liver transplantation from 2011 to 2020 after histologic examination and ultrasonography at Asan Medical Center (Seoul, South Korea) (IRB no. 2021-0839).
During the donor sample procurement, an intraoperative assessment of the liver was systematically carried out to rule out fibrosis, cirrhosis, steatosis, and other abnormalities before transplantation (Supplementary Table 1). Human liver samples of normal individuals (n = 5) or DILI patients (n = 22) were processed for RNA isolation, immunoblottings for Gα12, ALOX12, and qRT-PCR assays for miR-15a. All patients in this study provided written informed consent, and the study was approved by the institutional review board of Asan Medical Center.
For the human fibrosis analysis, nontumorous liver tissues adjacent to liver cancer were collected from patients who had been diagnosed with liver fibrosis or cirrhosis by histologic examination between 3 2006 and 2009 in Asan Medical Center (Seoul, Korea) (IRB no. 2012-0133) [1]. After resection, fresh surgical specimens were immediately snap-frozen in liquid nitrogen and stored at 80 ºC. Informed consent from the patients was obtained before operations and the study protocol was approved by the institutional review board of Asan Medical Center in accordance with the ethical guidelines of the 1975 Declaration of Helsinki. Ethics approval was provided by the ethics committees of Seoul National University. G protein subunit alpha 12 (Gα12), arachidonate 12-lipoxygenase (ALOX12), glutathione peroxidase 4 (GPX4), and microRNA-15a (miR-15a) levels were measured among normal (n = 2), portal fibrosis (n = 10), septal fibrosis (n = 15), and cirrhosis (n = 20) groups.

RNA seq analysis
For RNA-seq analysis, liver from either WT or Gna12 KO mice treated with APAP (300 mg/kg BW, i.p.) or vehicle were used (n = 3 each). The sampling protocol was applied in Gna12 KO mice and their age-matched WT littermates. Total RNA concentration was calculated by Quant-IT RiboGreen (Thermo Fisher Scientific, R11490). To assess the integrity of the RNA, samples were run on the Tape Station RNA screen tape (Agilent Technologies, 5067-5576). Only high-quality RNA preparations, with RIN N7.0, were used for RNA library construction. A library was independently prepared with 1 μg total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., RS-122-2101). The first step in the workflow involved purifying the poly-A containing mRNA molecules using poly-T-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen, 18064014) and random primers. This was followed by second-strand cDNA synthesis using DNA polymerase I, RNase H, and dUTP. These cDNA fragments then went through an end repair process, the addition of a single Adenovirus encoding mouse Gα12QL (Q229L) was kindly provided from Dr. Patrick J. Casey (Duke University Medical Center, Durham, NC). The mouse albumin enhancer/promoter construct, kindly provided by Dr. Richard D. Palmiter (University of Washington), was used to make a lentivirus encoding Gα12; the original elongation factor-1 promoter of pCDH-EF1-multiple cloning site-copepod super green fluorescent protein (copGFP) plasmid (System Biosciences) was replaced with the albumin enhancer/promoter. The coding region of pcDNA3-Gα12 was extracted and was cloned downstream of the albumin enhancer/promoter, as described previously [2]. The constructs were sequenced to assess the integrity of the insert. For in vivo experiments, 100 μl of 1.5×10 7 TU was administered to 10-weekold WT or Gna12 KO mice through the tail vein. At 7 days after injection, the mice were fasted overnight prior to a single dose of APAP treatment, and tissue samples were acquired 6 h later.

Cell lines and primary hepatocytes
AML12 cells (a mouse hepatocyte-derived cell line) were purchased from American Type Culture Collection (ATCC) (Rockville, Maryland), and were cultured in the DMEM/F-12 medium containing 10% FBS, insulin-transferrin-selenium X (ITSX), dexamethasone (40 ng/ml; Sigma), and the antibiotics. The cells with less than 20 passage numbers were used. Primary hepatocytes were isolated from C57BL/6 mice under the guidelines of the institutional animal use and care committee, as described previously [3], and plated in a 6-well dish at a density of 2 x 10 5 cells/well, and wells with 70% to 80% confluence were used. Briefly, under anesthesia with Zoletil, livers were perfused with Ca 2+ -free Hank's buffered salt solution (Invitrogen, Carlsbad, CA) for 10 min, followed by continuous perfusion with a 0.1% w/v collagenase (Sigma, Type I). The whole liver was removed, and minced in the phosphate-buffered saline. Mouse hepatocytes were filtered through a 0.2 µm cell strainer (BD Biosciences) and centrifuged at 50 g for 2 min (three times) to separate hepatocytes. Hepatocytes were harvested into collagen-coated plates in isolation media (Dulbecco's modified Eagle's medium

RNA isolation and quantitative RT-PCR assays
Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) and was reverse-transcribed. The resulting cDNA was amplified by qRT-PCR as previously described [1]. β-actin or 18S rRNA or GAPDH was used as the normalization control. qRT-PCR assays for miRNA, cDNA was generated from equal amounts of total RNA per sample (1 μg) using the miScript Reverse Transcription kit

Adenoviral infection
Adenovirus encoding for mouse Gα12QL (Q229L) was kindly provided by Dr. Patrick J. Casey (Duke University Medical Center, Durham, NC). For in vitro assays using adenovirus, Ad-GFP was used as infection control.

Immunoblot analysis
Cells were centrifuged at 3,000 g for 3 min and allowed to swell after the addition of lysis buffer in ice for 1 h. The lysates were centrifuged at 10,000 g for 10 min to obtain supernatants. Proteins were separated by 6%, 7.5%, or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto nitrocellulose membranes (Millipore, Bedford, MA). The membrane was blocked with 5% non-fat dried milk in Tris-buffered saline and Tween 20 (TBST) (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) for 1 h, and incubated overnight with primary antibodies at 4ºC. After washing with TBST buffer, membranes were incubated with a horseradish peroxidase-conjugated antimouse IgG secondary antibody for 1 h at room temperature. Bands were visualized using the ECL chemiluminescence system (GE Healthcare, Buckinghamshire, UK). Equal loading of proteins was verified by immunoblotting for β-actin. Quantifications were done by scanning densitometry of the immunoblots and β-actin normalization.

Immunohistochemistry
Formalin-fixed, paraffin-embedded human fibrotic liver samples were cut using a microtome into 4-μm thickness. The tissue sections were mounted on glass slides. Antibodies of interest (Gα12, ALOX12, and GPX4) were incubated overnight at room temperature with the slide-mounted method.
Mouse liver specimens were fixed in 10% formalin, embedded in paraffin, cut into sections, and were mounted on slides. Tissue sections were immunostained with the antibodies directed against Gα12, GRP78, 4-HNE, ALOX12, and GPX4. Briefly, the paraffin-embedded sections were deparaffinized with xylene and rehydrated with alcohol series. After antigen retrieval was performed, the endogenous peroxidase activity was quenched. The sections were pretreated with 10% normal donkey serum for 40 min to block nonspecific antibody binding and incubated with the antibodies of interest overnight at 4 ℃. The sections were then treated with 2% normal donkey serum for 15 min and incubated with biotin-SP-conjugated affinity pure donkey anti-rabbit IgG for 2 h. The labeling was done by using 3,3'diaminobenzidine. After mounting with Permount solution, the sections were examined using a light microscope (DMRE, Leica Microsystems, Wetzlar, Germany), and images were acquired with Fluoview-II (Soft Imaging System GmbH, Muenster, Germany) attached on the microscope.

Alox12 3'-UTR reporter assays
The miRNA 3'-untranslated region (UTR) target clone (Luc-Alox12-3'UTR), which contains Renila luciferase as internal control fused downstream to firefly luciferase, was purchased from 9 GeneCopoeia (MmiT090956-MT06; Rockville, MD). Luciferase activity assays were done according to manufacturer protocols. Briefly, AML12 cells were seeded in 6-well plates, and co-transfected with Luc-Alox12-3'UTR reporter and miR-15a mimic (or ASO) or its relative control using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA). The target site within the Alox12 3'UTR reporter was used to generate a mutant reporter construct (5'-UCGCCGCG-3', bolds indicate mutations), which was used as a negative control. Firefly and Renilla luciferase activities were measured sequentially using the Luc-Pair miR Luciferase Assay kit (GeneCopoeia, Rockville, MD). The activities were normalized with Renilla luciferase activities and expressed in relative luciferase activity units.

Blood biochemical analysis and histopathology
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were analyzed using Spectrum (Abbott Laboratories, Abbott Park, IL). Hematoxylin and eosin (H&E) staining was done as described [5]. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays were carried out using the in situ S7100 ApopTAG apoptosis detection kit from Millipore (Temecula, CA) and the DeadEnd Colorimetric TUNEL System from Promega (Madison, WI) according to the manufacturer's instructions. For the counting of TUNEL-positive cells, three fields/slides at ×100 magnification were randomly selected, and the percentage of TUNEL-positive staining was assessed using Image J (National Institutes of Health) software. Data were obtained from two to three samples per condition.

Flow cytometric analysis
Apoptosis was analyzed by the FITC-annexin V plus PI staining method according to the published method [6]. Briefly, cells were harvested by trypsinization. After washing with PBS containing 1% FBS, the cells were stained with 5 μl FITC-annexin V and 1 μl PI/~2ⅹ10 5  For E, values were expressed as mean ± SD (*P < 0.05, **P < 0.01). Statistical significance was tested via two-tailed Student's t-test or Pearson correlations. All values were expressed as mean ± SD (**P < 0.01). Statistical significance was tested via twotailed Student's t-test or one-way ANOVA coupled with Tukey's HSD multiple comparison procedure where appropriate. (E) Flow cytometric analyses for fluorescein isothiocyanate-annexin V and propidium iodide in AML12 cells treated with Tm (2 μg/ml, 12 h) 6 h after infection with ad-GFP or ad-Gα12.
For B-D, values were expressed as mean ± SD (*P < 0.05, **P < 0.01). Statistical significance was tested via one-way ANOVA coupled with Tukey's HSD or the LSD multiple comparison procedures where appropriate.