Abstract
Hepatic stem/progenitor cells are the major cell compartment for tissue repair when hepatocyte proliferation is compromised in chronic liver diseases, but the expansion of these cells increases the risk of carcinogenesis. Therefore, it is essential to explore the pathways restricting their expansion and abnormal transformation. The ligand of glucocorticoid-induced tumour necrosis factor receptor (GITRL) showed the most highly increased expression in hepatic progenitor cells treated with transforming growth factor (TGF)-β1. If overexpressed by hepatic progenitor cells, GITRL stimulated cell proliferation by activating the epithelial–mesenchymal transition pathway and enhancing ERK1/2 and Akt phosphorylation via GITRL binding to ANXA2. However, GITR, the specific GITRL receptor, suppressed the epithelial–mesenchymal transition pathway of GITRL-expressing cells and decreased their growth by dissociating ANXA2 from GITRL and reducing downstream ERK1/2 and Akt phosphorylation. This study identifies GITR/GITRL reverse signalling as a cross-interaction pathway between immune cells and hepatic stem/progenitor cells that restricts the expansion of hepatic stem/progenitor cells and reduces the possibility of carcinogenesis.
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Introduction
The liver is an organ with a strong regenerative capacity in response to injuries that involve hepatocytes and facultative stem/progenitor cells [1, 2]. Acute and short-term liver injuries stimulate existing hepatocytes to replicate for injury repair and cell replenishment. However, when hepatocyte proliferation is compromised in chronic liver diseases, hepatic stem/progenitor cells, resident in the portal area, serve as a major cell compartment for tissue repair and liver regeneration [3]. The number of hepatic stem/progenitor cells is increased as chronic disease progresses, and the extent of hepatocytes derived from hepatic stem/progenitor cells is correlated with disease severity [4, 5]. However, given that severe chronic liver diseases enhance the incidence of hepatocellular carcinoma (HCC) [6, 7] and that HCC shares many common markers with hepatic stem/progenitor cells [8, 9], it has been postulated that hepatic stem/progenitor cells are a possible cellular origin of HCC. Therefore, modulating the expansion or proliferation of hepatic stem/progenitor cells and preventing their abnormal transformation are essential for balancing regeneration and carcinogenesis.
Chronic hepatitis results from repeated cycles of hepatocyte death, inflammation, and regeneration. Dead hepatocytes not only activate liver resident immune cells, such as Kupffer cells, but also recruit many immune cells, including neutrophils, monocytes and T cells, to the liver tissue [10, 11]. It has become evident that the inflammatory microenvironment produced by recruited monocytes [12], macrophages [13], T cells [14, 15] and NK cells [14, 15] contributes to the expansion of hepatic stem/progenitor cells in liver injury and regeneration models. Cytokines secreted by these immune cells, including IL-22 [16], TNF [17, 18], TNF-like weak inducer of apoptosis (TWEAK) [19,https://david-d.ncifcrf.gov/for functional annotation for signalling pathway analysis.
GSEA for RNA array or RNA sequencing data
GSEA was performed by the R/clusterProfiler package by annotating rat gene sets to predefined human gene sets from the Molecular Signatures Database [55]. A list of ranked genes from RNA array data with GEO accession number GSE165858 was used for GSEA to compare the TGF-β1-treated cells to the control cells [56]. A list of ranked genes from the RNA sequencing data of PRJNA698431 was used for fast GSEA performed by the R/Bioconductor package to compare the IgG-treated GITRL-overexpressing cells to the IgG-treated vector-transfected control cells [57].
Cell growth and proliferation analysis
Cell growth and proliferation were analysed by growth curves and the Ki-67-positive rate as described previously [54]. For growth curves, a total of 3 × 103 cells were plated in triplicate in antibiotic-free complete medium in E-Plate 16 (ACEA Biosciences, San Diego, CA, USA) on the xCELLigence Real-Time Cell Analyzer (RTCA)-MP system (ACEA Biosciences) according to the manufacturer’s instructions. The cell index (CI) was read automatically and continuously recorded every hour as CI ± SD. Growth stimulation was determined at the time of maximum cell index (CImax), and a non-parametric t test was used to analyse the significant differences in cell growth. For Ki-67-positive rate analysis, the investigator was blinded to the samples, and ImageJ 1.51j8 (National Institute of Health, USA) was used for analysis.
Western blot
Protein extracts were prepared and analysed by western blots according to standard protocols as described previously [51] using primary antibodies, including ERK1/2 (1:2000, Cell Signaling Technology), phospho-p44/42 MAPK [ERK1/2(Thr202/Tyr204), 1:2000, Cell Signaling Technology], Akt (1:2000, Cell Signaling Technology), phosphor-Akt(Ser473) (1:2000, Cell Signaling Technology), and ANXA2 (1:2000, Cell Signaling Technology). Bands were detected using the Molecular Imager ChemiDoc XRS + with Image Lab Software version 3.0 (Bio-Rad, Hercules, CA, USA).
Immunoprecipitation (IP) and mass spectrometry analysis
Immunoprecipitation was carried out according to the manufacturer’s instructions of the PierceTM MS-compatible Magnetic IP kit (Protein A/G, Thermo, Rockford, IL, USA). Briefly, 1.0 × 107 cells were washed with PBS once and incubated with ice-cold IP-MS cell lysis buffer on ice for 10 min with periodic mixing. After centrifugation at 13,000×g for 10 min, the supernatant was transferred to a new tube for protein quantification. Then, 10 μl of His-tag antibodies (1:100, Cell Signaling Technology), or 2 μg GITRL antibodies (Invitrogen) or 2 μg control IgG (Sigma-Aldrich, St Louis, MO, USA) was incubated with cell lysate at 4 °C overnight. Prewashed magnetic beads were added to the sample/antibody mixture for incubation at room temperature for 1 h with agitation. After collection by the magnetic stand and washing with IP-MS Buffer A three times and IP-MS Buffer B twice, the beads were eluted by IP-MS elution buffer at room temperature for 10 min. Finally, the elution buffer was dried with a speed vacuum concentrator D-AQ (Eppendorf, Germany) for 2 h, and the pulldown proteins were resuspended in protein lysis buffer. After centrifugation at 1200 rpm for 2 min, the supernatant was used for SDS-PAGE analysis, and the protein bands revealed by silver staining were cut out for mass spectrometry analysis (BGI).
Statistical analysis
Sample sizes were chosen based on previous similar experimental outcomes. Data are presented as the mean value ± SD and were analysed for significance using non-parametric t tests by GraphPad Prism 6 software (GraphPad Software, Inc., CA, USA). P < 0.05 indicated a significant difference.
Data availability
RNA array data (GEO accession number: GSE165858) and RNA sequencing data (NCBI Sequence Read Archive accession number: PRJNA698431) used during the study are available online in accordance with funder data-retention policies.
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Acknowledgements
We would like to thank the Clinical Data and Biobank Resource of Bei**g Friendship Hospital for storing the liver samples. We would also like to thank Ms. **ng Jia from Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Sciences for her help in taking and analysing 3D-SIM images.
Funding
This work was supported by the Chinese Foundation for Hepatitis Prevention and Control of the Wang Baoen Liver Fibrosis Foundation (2019073), the National Natural Science Foundation of China (81570548, 81870399 and 81770598), and the National Science and Technology Major Special Project for New Drug Development (Grant number 2018ZX09201016).
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PW, DZ and JJ conceived the study and the experimental design and supervised the study. YH, YP, KL, LL, YT and HL performed the experiments and acquired the data. MC, TL, HM and HY helped perform the experiments and edit the manuscript. PW, DZ and JJ wrote and edited the manuscript. All authors read and approved the final manuscript.
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The use of frozen human liver tissues in the Clinical Data and Biobank Resource of Bei**g Friendship Hospital was approved by the Ethics Committee of Bei**g Friendship Hospital, Capital Medical University (No. 2018-P2-055-01, Bei**g, China).
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He, Y., Pei, Y., Liu, K. et al. GITR/GITRL reverse signalling modulates the proliferation of hepatic progenitor cells by recruiting ANXA2 to phosphorylate ERK1/2 and Akt. Cell Death Dis 13, 297 (2022). https://doi.org/10.1038/s41419-022-04759-z
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DOI: https://doi.org/10.1038/s41419-022-04759-z
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