Ceramide kinase-mediated C1P metabolism attenuates acute liver injury by inhibiting the interaction between KEAP1 and NRF2

Acute liver injury is the basis of the pathogenesis of diverse liver diseases. However, the mechanism underlying liver injury is complex and not completely understood. In our study, we revealed that CERK, which phosphorylates ceramide to produce ceramide-1-phosphate (C1P), was the sphingolipid pathway-related protein that had the most significantly upregulated expression during acute liver injury. A functional study confirmed that CERK and C1P attenuate hepatic injury both in vitro and in vivo through antioxidant effects. Mechanistic studies have shown that CERK and C1P positively regulate the protein expression of NRF2, which is a crucial protein that helps maintain redox homeostasis. Furthermore, our results indicated that C1P disrupted the interaction between NRF2 and KEAP1 by competitively binding to KEAP1, which allowed for the nuclear translocation of NRF2. In addition, pull-down assays and molecular docking analyses revealed that C1P binds to the DGR domain of KEAP1, which allows it to maintain its interaction with NRF2. Importantly, these findings were verified in human primary hepatocytes and a mouse model of hepatic ischemia‒reperfusion injury. Taken together, our findings demonstrated that CERK-mediated C1P metabolism attenuates acute liver injury via the binding of C1P to the DGR domain of KEAP1 and subsequently the release and nuclear translocation of NRF2, which activates the transcription of cytoprotective and antioxidant genes. Our study suggested that the upregulation of CERK and C1P expression may serve as a potential antioxidant strategy to alleviate acute liver injury.


Clinical Specimens
Human liver tissues of hepatic laceration (n=5) were obtained from the Affiliated Hospital of Guilin Medical University (Guilin, China).The patients with a hepatic laceration underwent liver segment resection.Clinical samples were collected from patients after obtaining informed consent in accordance with a protocol approved by the Ethics Committee of the Affiliated Hospital of Guilin Medical University (Guilin, China).

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from liver tissues or cell lines with TRIzol (TAKARA, Japan).Then, the RNA was reverse-transcribed to cDNA.qRT-PCR was performed according to the manufacturer's instructions on a 7500 Real-Time PCR System (Thermo Fisher, MA, USA) using TB Green Premix Ex Taq II (TaKaRa, Dalian, China).The relative expression of the target genes was normalized to that of the control, GAPDH, using the 2 -ΔΔCt method.

Western blotting
Total protein was extracted from liver tissues or cells by RIPA buffer (Solarbio, China) containing a protease inhibitor.The cytoplasmic and nuclear proteins were extracted following the instructions of the cytoplasmic and nuclear fractionation kit for cells (SC-003, Beijing, China) or for frozen/fresh tissues (NT-032, Invent, China).The extracted proteins were boiled, loaded onto, separated with 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, and transferred to polyvinylidene difluoride membranes.The membranes were subsequently blocked in TBS-Tween with 5% skim milk for 1 h at room temperature.Then, the membranes were incubated overnight at 4°C with primary antibodies.After washing, the membranes were incubated for 1 h at room temperature with an HRP-conjugated secondary antibody (Ray, China) and then incubated with a Super ECL detection reagent (Yeasen, China).The immunoreactive bands were visualized by a Tanon Automatic Chemiluminescence and Fluorescence Image Analysis System (Tanon, China).

Immunohistochemistry (IHC)
After drying, dewaxing, and rehydration, paraffin-embedded liver tissue sections were subjected to high-pressure antigen repair with citrate buffer (0.01 M, pH 6.0) and were subsequently incubated in 0.3% H 2 O 2 solution for 10 min to block endogenous peroxidase activity.Then, the sections were blocked in PBS-Tween with 5% BSA for 1 h and incubated with rabbit polyclonal anti-CerK antibody (1:200) at 4°C overnight.Then, the sections were incubated with a second antibody for 1 h and stained with 3,3'-diaminobenzidine tetrahydrochloride (DAB) and then hematoxylin.Then, the sections were dehydrated, cleared, sealed with coverslips, and analyzed by microscopy.

Immunofluorescence
A total of 4×10 4 cells were plated in 24-well plates for 24 h.After treatment, the cells were fixed with 4% PFA for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, incubated with a rabbit anti-NRF2 antibody and mouse anti-KEAP1 antibody, incubated with Alexa-conjugated secondary antibodies, and finally stained with DAPI.Images were acquired by a Nikon AXR confocal microscope.

Enzyme-linked immunosorbent assay (ELISA)
The C1P levels in tissues or cell lysates were quantified using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol.

Cell treatments
For C1P treatment, cells were treated with 0.4 mM H 2 O 2 and different concentrations of C1P for 24 h.For NVP-231 treatment, cells were treated with 0.4 mM H 2 O 2 and different concentrations of NVP-231 for 24 h.For C1P supplementation assays in shRNA-CERK cells, control and shCERK cells were treated with 0.4 mM H 2 O 2 and 20 μM C1P for 24 h.For 4-Octyl Itaconate (OI) treatment, cells were treated with 0.4 mM H 2 O 2 and C1P or NVP-231 for 24 h.After 12 h, cells were treated with 62.5 μM 4-OI for 12 h.