FoxO3 restricts liver regeneration by suppressing the proliferation of hepatocytes | npj Regenerative Medicine – Nature.com

Cell culture

NCTC1469, a liver cell line established in the 1957 from a neonatal mouse36, was obtained from the FuDan IBS Cell Center (FDCC, Shanghai, China). NCTC1469 cells were cultured in Dulbeccos modified Eagles medium (DMEM) (Corning, USA) supplemented with 10% (v/v) Horse serum (Hyclone, Logan, UT, USA), 100 units/ml penicillin and 100g/ml streptomycin (Gibco, USA), at 37C in a 5% CO2 incubator. Culture medium was replaced every 23days and cells were passaged when they reached 80% confluence. Cells were mycoplasma negative through treatment with LookOut Mycoplasma Elimination Kit (Sigma-Aldrich). Gene silencing was achieved by transfecting predesigned siRNA duplexes (Supplementary Table 3) designed and synthesized by RiboBio (Guangzhou, China).

Stable knockdown of the FoxO3 gene in NCTC1469 cells was achieved by lentiviral based short-hairpin RNA delivery37. FoxO3 specific shRNA or negative control were cloned into pLOX-U6-Puro vectors for viral particle package in HEK 293T cells. Infected NCTC1469 cells were selected by puromycin and expanded to form a stable sub-line. Knockdown efficiency was confirmed at both mRNA and protein levels. The shRNA sequences are as follows: FoxO3-shRNA: 5-GGA ACT TCA CTG GTG CTA AGC-3; negative shRNA (NC): 5-ACT ACC GTT GTT ATA GGT G-3. Stable cell line generated by FoxO3-shRNA and negative shRNA were named as shFoxO3 cells and shNC cells, respectively. To establish FoxO3 overexpression stable cells, NCTC1469 cells were transfected by pLOX-FoxO3 lentiviruses19 and screened by puromycin and expanded to form a stable sub-line.

For cell proliferation assay, NCTC1469 cells were incubated for 48h in 24 well plates with different treatments. DNA synthesis was then analyzed by 5-Ethynyl-2'-deoxyuridine (EdU) labeling, using Cell-Light EdU Apollo567 In Vitro Imaging Kit (RiboBio, Guangzhou, China) according to the manufacturers instructions. At least seven images were randomly taken per well using a Zeiss LSM 700 laser confocal microscope (Carl Zeiss). The population of EdU+ cells was determined by counting at least 500 cells per well. The EdU+ cells were quantified as the percentage of total cells. In addition, cell proliferation was also analyzed by evaluating cell count using the Enhanced Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, China) according to the manufacturers instructions.

Total RNA was isolated using RNeasy kit (Qiagen, Valencia, CA, USA) from NCTC1469 cells according to the manufactures instruction. The cDNA was transcribed from total RNA using SuperScript III Reverse Transcriptase (Roche, USA). The coding sequence (CDS) of Nox4 (NM_015760.5) was amplified by the KOD-Plus-Neo Kit (Toyobo, Japan) and cloned into the pcDNA3.1 expression vector to construct the pcDNA-Nox4 plasmid. Constructed overexpression vector pcDNA-Nox4 and control plasmid pcDNA3.1 (pcDNA) were transfected into the FoxO3-overexpressing NCTC1469 cells to explore the effect of target gene on FoxO3-mediated proliferation and apoptosis.

FoxO3-LoxP-targeted (FoxO3fl/fl) mice (C57BL/6 background) were created by Cyagen Biosciences (Suzhou, China)19. The exon 3 region of the FoxO3 gene was flanked by LoxP sites and deleted upon Cre-mediated recombination (Fig. 2a). The Albumin (Alb)-Cre transgenic line was obtained from the Jackson Laboratory (stock number 003574). Alb-Cre mice were genotyped using oIMR5374 (GAA GCA GAA GCT TAG GAA GAT GG) and 20240 (TTG GCC CCT TAC CAT AAC TG) primers and identified by a 390bp PCR product. For Alb-Cre mice, Alb promoter drive Cre recombinase expression primarily in hepatocytes with variable expression in cholangiocytes depending on the floxed gene38,39. FoxO3fl/fl mice were intercrossed with Alb-Cre mice to generate FoxO3fl/+::Alb-Cre (FoxO3+/-, heterozygote). Heterozygotes were then crossed with FoxO3fl/fl mice to generate FoxO3fl/fl::Alb-Cre mice (FoxO3-/-, hereafter termed AKO) with constitutive loss of FoxO3 in hepatocytes. The FoxO3fl/fl mice obtained from the same breeding were used as control mice (Con). Age-matched FoxO3fl/fl mice were used as control (Con). To induce conditional knockout of FoxO3 in adult liver alone, adeno-associated virus serotype 8 (AAV8) was constructed to specifically express Cre recombinase in liver. To confirm the recombination efficiency of Cre, AAV8-Tbg-Cre (AAV-Cre) virus was intravenously injected into R26-CAG-LSL-EGFP mice (Shanghai Model Organisms Center, Shanghai, China) with different concentrations. Two weeks later, the percentages of EGFP+ liver cells were then checked to evaluate Cre recombination efficiency. We found that AAV-Cre adeno-associated virus with the concentration of 51011 gc/mouse has a highest efficiency (Supplementary Fig. 11). Therefore, AAV-Cre or negative control (AAV-NC) adeno-associated virus was injected intravenously at a concentration of 51011 gc/mouse in FoxO3fl/fl mice 2weeks prior to partial hepatectomy (PH). For knockdown of Nr4a1 in adult AKO mice, AAV8-mediate shRNA targeting for Nr4a1 (AAV-shNr4a1) or negative control (AAV-shNC) adeno-associated virus was injected intravenously at a concentration of 51011 gc/mouse in AKO mice 2weeks prior to partial hepatectomy (PH). To overexpress Nox4 in AKO mice, AAV8-Tbg-Nox4 (AAV-Nox4) or negative control (AAV-NC) adenovirus was injected intravenously at a concentration of 51011 gc/mouse in FoxO3-deficient mice 2weeks prior to liver injury. For experiments using adult animals, male mice were used in this study. All animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of Jinan University. Data and methods are reported here in accordance with ARRIVE guidelines40.

Male animals aged 8~9weeks were kept on a 12h-day/night cycle with free food/water access, and were used for partial hepatectomy (PH) experiments. During anesthesia induced by isoflurane, mice were subjected to 70% PH or 30% PH surgery. The surgery was performed between 8 and 12am to remove the median and left lobes and the mortality was <5%. The weight of regenerated livers as well as the whole body was measured at 07days post-PH (dpH). The ratio of wet weight of the remaining liver after PH over the weight of the whole animal was taken as the liver to body weight ratio. The 0 dpH denotes the time point in quiescent animal before PH. To induce another classic liver injury model, male adult mice were injected with CCl4 (0.6mL/kg, intraperitoneally) diluted in olive oil three times a week (every 48h) for 4weeks and euthanized 48h after the last injection as previously reported41.

Mouse livers were flushed via the portal vein using perfusion buffer containing Collagenase Type IV, followed by primary hepatocyte isolation and purification. Isolated and purified hepatocytes were fixed at 4 with 70% ethanol overnight at 4C, washed three time with PBS, and incubated with propidium iodide (PI, 50g/ml, Sigma)/RNAse A (ribonuclease, 1mg/ml, Sigma) solution for 3h at room temperature, followed by flow cytometry analysis with a FACS Aria cytometer (Becton Dickinson) to determine DNA content and ploidy distribution using ModFit LT 5.0 software (Verity Software House). Growth assay of primary hepatocytes were performed as previously report21. In brief, isolated hepatocytes were cultured in mouse hepatocyte growth medium (MHGM) with or without mitogens including EGF and HGF (Gibco, 40ng/ml for each) for 4days as described previously. DNA synthesis were then analyzed by 5-Ethynyl-2'-deoxyuridine (EdU) labeling, using Cell-Light EdU Apollo567 In Vitro Imaging Kit (RiboBio, Guangzhou, China) according to the manufacturers instructions.

Liver tissues were fixed in 4% formaldehyde and dehydrated in a series of ethanol. The fixed liver was embedded in paraffin and sectioned to 5m thickness. Sections were subjected to hematoxylin and eosin (H&E) staining for morphological and mitotic analysis. Paraffin-embedded liver sections were deparaffinized in xylene, followed by rehydration through a graded ethanol series and staining with hematoxylin and eosin stain (H&E). Mitotic activity in liver was examined and quantified as previously described2. In brief, mitotically active areas were first screened under lower magnification. For quantification, total mitotic counts in five high-magnification fields in the most mitotically active areas were considered for each mouse using a light microscope coupled with a digital image acquisition system.

Liver sections were subjected to immunofluorescence staining to analyze the expression of cell proliferating markers using a confocal fluorescence microscope42. In brief, livers were embedded in paraffin and cut in 5m sections, followed by deparaffinization and rehydration. Antigen retrieval were routinely performed using Citrate Antigen Retrieval Solution (Beyotime Biotechnology, P0081) according to the manufacturers instructions. Sections were permeabilized with 0.5% Triton X-100/PBS and then blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, USA) for 1h at room temperature, and incubated with appropriate primary antibodies overnight at 4C. After washing with PBS, sections were incubated with corresponding secondary antibodies conjugated to fluorescence for 1h at room temperature, followed by counterstaining with DAPI (Sigma). Secondary antibodies used are following: Alexa Fluor 488 donkey anti-rabbit IgG (Abcam, 1:500), Alexa Fluor 405 donkey anti-goat IgG (Abcam, 1:200), and Cy3-conjugated Affinipure Goat anti-rabbit IgG (Proteintech, 1:100). For immunohistochemistry staining, sections were stained with primary antibody overnight at 4C. SignalStain Boost IHC Detection Reagents (HRP, CST) and SignalStain DAB Substrate Kit (CST) were used for amplification and development, respectively. The detailed information of primary antibodies used in this study can be found in the Supplementary Table 4. For immunostaining in liver tissues, species isotype (Santa Cruz) was used as negative control to confirm the specificity of primary antibodies. Images were captured by laser-scanning confocal microscope (LSM 700, Zeiss) and analyzed by ZEN 2012 software (Zeiss). Given that FoxO3 was predominantly expressed in nuclei of liver cells (Fig. 1a, b), FoxO3 fluorescence intensity in nuclei were normalized to nuclei size (mean fluorescence intensity, MFI) over total slides using ZEN 2012 software (Zeiss) to evaluate the total expression levels of FoxO3 protein. For the quantification of cell proliferation, the proliferation of hepatocytes (HNF4+ cells) was quantified by the percentage of proliferating hepatocytes (Ki67+ HNF4+ cells, or pH3+ HNF4+ cells) relative to total hepatocytes (HNF4+ cells). For some experiments, the proliferation of non-hepatocytes (HNF4 cells) was quantified by the percentage of proliferating non-hepatocytes (Ki67+ HNF4 cells, or pH3+ HNF4 cells) relative to total liver cells (DAPI+ cells).

Hepatocyte size was evaluated by -catenin staining. In brief, following deparaffinized, rehydrated, slides were then incubated with anti--catenin (BD, 1:500) overnight at 4C, followed by a further incubation at room temperature for 1h with goat anti-rabbit IgG H&L (Alexa Fluor 488) preadsorbed secondary antibody. Nuclear DNA was labeled in blue with DAPI. To quantify the cell size, 5 independent livers per group (at least 300 cells) were captured with laser-scanning confocal microscope (LSM 700, Zeiss). ZEN 2012 lite software (Zeiss) was used to quantify the relative size of each cell. Moreover, the relative size of primary hepatocytes was also evaluated by the forward scatter (FSC) intensity in flow cytometry.

For EdU labeling experiments, mice were intraperitoneally injected with 100l of a 1mg/ml solution of EdU (RiboBio, Guangzhou, China) dissolved in PBS. Livers were embedded in Tissue-Tek optimal cutting temperature compound (OCT) (Sakura, USA) for frozen section (4 m). Sections were rinsed three times in PBS and fixed in 4% parapormaldehyde for 30min. After rinsing three times again, citrate antigen retrieval was performed as described above. Sections were then incubated with 2mg/mL glycine solution for 10min, permeabilized with 0.5% Triton X-100 in PBS for 10min, and then rinsed with PBS once for 5min. This was followed by incubation with Apollo576 staining solution (1) at room temperature for 30min. Sections were washed three times in PBS, stained with DAPI for 10min to label nuclei, and mounted in Antifade Mounting Medium. Images were captured by a laser-scanning confocal microscope (LSM 700, Zeiss) and analyzed by ZEN 2012 software (Zeiss).

To analyze hepatocyte proliferation at the indicated time points, EdU was injected 2h prior to liver collection. For EdU pulse-chase experiments, EdU was multiply injected at indicted time points to label all proliferating hepatocytes during the whole period of liver regeneration. The last injection was performed 2h prior to liver collection. Sham-operated mice underwent the same procedure without the liver resection. For the quantification of cell proliferation, the proliferation of hepatocytes (HNF4+ cells) was quantified by the percentage of proliferating hepatocytes (EdU+ HNF4+ cells) relative to total hepatocytes (HNF4+ cells). But the proliferation of non-hepatocytes (HNF4- cells) was quantified by the percentage of proliferating non-hepatocytes (EdU+ HNF4- cells) relative to total liver cells (DAPI+ cells).

Blood was collected by shearing the right atrium and was allowed to coagulate for 2h on ice. Serum was the isolated as the supernatant fraction after centrifugation at 2000rpm for 5min. Hepatic function was analyzed using serum ALT and AST activities, which are markers of injury. Serum ALT and AST activities were determined using the Hitachi 7600 automatic biochemical analyzer (Japan) and were expressed as units per liter (U/L). In addition, enzyme activities in liver tissue were determined using commercial kits produced by Jiancheng Institute of Biotechnology (Nanjing, China). Tissue enzyme activities were normalized to total protein weight and were expressed as units per gram protein (U/g).

For total RNA isolation, liver tissues were extracted in sham and injured mice at 4days post-PH (dpH), respectively. Three mice per group were used for RNA-sequencing analysis. RNA preparation, library construction and sequencing on GBISEQ-500 platform was performed. After filtering the reads with low quality, clean reads were then obtained and mapped to the reference genome of mouse (GRCm38.p6) with the HISAT43. Gene expression level was quantified by a software package called RSEM44 and expressed as fragments per kilobase of exon per million fragments mapped (FPKM). Differential expressed (DE) genes were detected using NOISeq method45 with Probability0.8 and fold change (FC) of FPKM2. Only those genes were considered for the differential expression analysis, which displayed FPKM1 in either of the two samples under comparison. GO analysis was performed using online tool DAVID 6.8 (https://david.ncifcrf.gov/summary.jsp), and terms with p-value0.05 were included. Differentially expressed gene heat maps were clustered by hierarchical clustering using cluster software46. RNA sequencing data have been deposited in NCBI Sequence Read Archive (SRA) under the accession code SRP224045.

Total RNA was isolated using RNeasy kit (Qiagen, Valencia, CA, USA) from cells or liver tissue according to the protocol of the manufacturer, respectively. Reverse transcription to cDNA was performed with 30ng of total RNA, random primers, and SuperScript III Reverse Transcriptase (Roche, USA). The qPCR was performed using a Light Cycler 480 SYBR Green I Master (Roche, USA) and the MiniOpticon qPCR System (Bio-Rad, CA, USA). After denaturation for 10min at 95C, the reactions were subjected to 45 cycles of 95C for 30s, 60C for 30s, and 72C for 30s. GAPDH was used as the internal standard control to normalize gene expression using the 2Ct method. The sequences of the qPCR primers were listed in Supplementary Table 5.

Tissues or cells were lysed in RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Sigma) for SDS-PAGE. Protein concentrations were determined using Bio-Rad Protein Assay (Bio-Rad Laboratories). 30g of protein were separated by SDA-PAGE, proteins were transferred onto PVDF membranes (Millipore), then blocked in 5% nonfat milk/TBS-Tween 20 and incubated with primary antibodies (dilution in TBST) overnight at 4C. Membranes were then washed and incubated with corresponding second antibodies for 1h at room temperature. Bands were detected by chemiluminescence reagents (ThermoFisher Scientific). Primary antibodies can be found in the Supplementary Table 4. Secondary antibodies used are following: goat-anti-mouse horseradish peroxidase (HRP)-conjugated antibody (CST, 1:3000) and goat-anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (CST, 1:2000). Regarding the visualization of housekeeping protein (-actin), the partial membrane was cropped from the same one as the target proteins of interest when their molecular weights are greatly different from each other. Otherwise, two members with same loading dose of total protein were simultaneously performed to visualize the target protein and -actin, respectively. Given that stripping and re-probing may result in errors between different bands, stripping buffer was not used in this study. Chemiluminescent signals were quantitated with Image-Pro Plus version 6.0 software (Media Cybernetics, MD, USA). The expression level of target protein was set as 100% in control group. Relative expression levels of target protein in experimental groups were expressed as the percentage of control group. All blots derive from the same experiment and were processed in parallel. The uncropped blots are listed in the Supplementary Figs. 24 and 25.

The promoter sequences (2000bp to 1bp, upstream of TSS) of the mouse Nox4 (Gene ID: 50490) and Nr4a1 (Gene ID: 15370) genes were analyzed by JASPAR 2018 online software (http://jaspar.genereg.net/)47 to determine potential FoxO3 binding sites (Supplementary Tables 1 and 2). The predicted binding site with highest score for target gene promoters were further analyzed by luciferase reporter assay, to evaluate the regulation effects of FoxO3 on the expression of mouse Nox4 and Nr4a1 genes. The pCMV-Gaussia-Dura Luc and pTK-Red Firefly Luc plasmids from ThermoFisher were used to construct the dual-luciferase reporter plasmid (pGL-RF) (Supplementary Figs. 19b and 22b), which contained both a Gaussia luciferase (GL) and a red firefly luciferase (RF). The predicted binding sites with highest score for target gene promoters were further analyzed by pGL-RF luciferase dual reporter assay, to evaluate the in vitro binding and regulating effects of FoxO3 on Nox4 and Nr4a1 genes. Briefly, promoter regions of target genes comprising predicted FoxO3 binding sites were amplified and cloned into the pGL-RF plasmid. NCTC1469 cells were co-transfected with dual reporter plasmids and pcDNA-FoxO3 plasmid (50ng for each plasmid) using LipoFiter Liposomal Transfection Reagent (Hanbio Biotechnology). Two days after transfection, luciferase reporter assay was carried out using the Pierce Gaussia-Firefly Luciferase Dual Assay Kit (ThermoFisher Scientific) according to the manufacturers protocol. Luciferase activity was measured using a BioTek SynergyTM 4 multimode microplate reader (BioTek Instruments). The relative activity of the Gaussia luciferase (GL) was normalized by the activity of red firefly luciferase (RL) and was expressed as fold change of control group. To further verify the potential binding sites, mutant luciferase reporter plasmids were generated by KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan), according to the manufacturers protocol.

ChIP assays were performed to evaluate the in vivo binding of FoxO3 to its consensus sequence in mouse Nr4a1 and Nox4 promoters. The assays were performed in mouse liver tissue using the SimpleChIP Plus Enzymatic Chromatin IP Kit (CST, #9004) according to the manufacturers instructions. FoxO3 antibody (CST, #2497) was used to immunoprecipitation assay. Normal goat IgG (CST, #2729) was used as a control as previously described. The DNA isolated from input chromatin fragments and from the precipitated chromatin fragments by anti-FoxO3 antibody or control IgG was subjected to PCR using primers flanking the consensus FoxO3 binding sites on Nox4 promoter. PCR products were determined on a 1.5% agarose gel. Relative binding ability of FoxO3 was expressed as the DNA signals relative to input. ChIP-PCR primers used in this study as follows: Nr4a1-ChIP-F: 5'-GGC CTC ACT TTT TCC ACC TAG T-3' and Nr4a1-ChIP-R: 5'-CCA GGG TAG GGT TGC TGT TTC-3'; Nox4-ChIP-F: 5'-TTG ACT TTG CAA TTA GCA GTA-T-3' and Nox4-ChIP-R: 5'-AGT CAG AAG CCC AAG TCT TCC T-3'.

All statistics were calculated using GraphPad Prism 8 Software. Among three or more groups, statistical analysis was performed using one-way or two-way ANOVA with Dunnetts multiple comparisons post hoc tests. Comparisons between two groups were analyzed using unpaired and 2-tailed Students t-test. All data are presented as the meanSEM. A p-value<0.05 was considered statistically significant. In this study, statistical analysis was in accordance with homogeneity of variance and normality of residuals. Homogeneity of variance was evaluated by the F-test with a p-value of >0.05. Normality of residuals was evaluated by D'AgostinoPearson test and/or ShapiroWilk test with a p-value of >0.05.

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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FoxO3 restricts liver regeneration by suppressing the proliferation of hepatocytes | npj Regenerative Medicine - Nature.com