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. 2018 Sep 6;33(2):1824–1835. doi: 10.1096/fj.201800736R

Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation

Chengcheng Ji *,†,1, Katsuya Nagaoka *,1, Jing Zou *,‡,1, Sarah Casulli *, Shaolei Lu §, Kevin Y Cao *, Hongyu Zhang *, Yoshifumi Iwagami *, Rolf I Carlson *, Keri Brooks *, Jonathan Lawrence *, William Mueller *, Jack R Wands *, Chiung-Kuei Huang *,2
PMCID: PMC6338639  PMID: 30188753

Abstract

The 5-hydroxymethylcytosine (5hmc) is a newly identified epigenetic modification thought to be regulated by the TET family of proteins. Little information is available about how ethanol consumption may modulate 5hmC formation and alcoholic liver disease (ALD) progression. A rat ALD model was used to study 5hmC in relationship to hepatocyte apoptosis. Human ALD liver samples were also used to validate these findings. It was found that chronic ethanol feeding significantly reduced 5hmC formation in a rat ALD model. There were no significant changes in TET2 and TET3 between the control- and ethanol-fed animals. In contrast, methylcytosine dioxygenase TET1 (TET1) expression was substantially reduced in the ethanol-fed rats and was accompanied by increased hepatocyte apoptosis. Similarly, knockdown of TET1 in human hepatocyte–like cells also significantly promoted apoptosis. Down-regulation of TET1 resulted in elevated expression of the DNA damage marker, suggesting a role for 5hmc in hepatocyte DNA damage as well. Mechanistic studies revealed that inhibition of TET1 promoted apoptotic gene expression. Similarly, targeting TET1 activity by removing cosubstrate promoted apoptosis and DNA damage. Furthermore, treatment with 5-azacitidine significantly mimics these effects, suggesting that chronic ethanol consumption promotes hepatocyte apoptosis and DNA damage by diminishing TET1-mediated 5hmC formation and DNA methylation. In summary, the current study provides a novel molecular insight that TET1-mediated 5hmC is involved in hepatocyte apoptosis in ALD progression.—Ji, C., Nagaoka, K., Zou, J., Casulli, S., Lu, S., Cao, K. Y., Zhang, H., Iwagami, Y., Carlson, R. I., Brooks, K., Lawrence, J., Mueller, W., Wands, J. R., Huang, C.-K. Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

Keywords: epigenetic modification, DNA methylation, 2-oxoglutarate, Krebs cycle, 5-azacytidine

Liver cirrhosis is one of the leading causes of death in the world (1, 2). An untreated fibrotic liver that caused by infection with hepatitis B or C, nonalcoholic steatohepatitis, or alcoholic liver disease (ALD) might well advance to cirrhosis (3). ALD accounts for half of the deaths associated with liver cirrhosis. Currently, there is no effective treatment available for patients with a cirrhotic liver. Liver transplantation is probably the most effective treatment option (4, 5). However, patients often die before transplantation. Thus there is an urgent need to develop effective therapies for liver cirrhosis patients. Understanding disease progression will advance our knowledge of its pathogenesis and could lead to the development of new preventative and therapeutic measures against it.

Several epigenetic events, including microRNA, DNA methylation, histone acetylation, and histone methylation, have been shown to participate in the progression of ALD (6–9). Hypomethylation of DNA in the liver in particular is implicated. In contrast, DNA hypermethylation has been found to occur in the circulating genomic DNA of ALD patients (10, 11). Recently identified 5-hydroxymethylcytosine (5hmC) in mammalian DNA represents a new type of epigenetic factor (12). The 5hmC modification is regulated by the members of the TET family of enzymes, including methylcytosine dioxygenase TET1 (TET1), TET2, and TET3 (13–15), and the enzymatic reaction is dependent on cosubstrates, including oxygen, iron, and 2-oxoglutarate. It has been shown that 5hmC levels are decreased in different liver fibrosis models through down-regulation of TET2 and TET3 expression (16) because RT-PCR analysis reveals that TET1 mRNA is not detectable in the livers of experimental animals (16). However, by using RNA sequencing techniques of the Genotype-Tissue Expression (GTEx) project, TET1 is detectable in human liver. Although 5hmC levels are down-regulated in ALD samples, there is no significant change in the expression of TET2 and TET3 (16). Interestingly, other studies have shown that TET1 protein is expressed in normal mouse and normal human livers (17, 18). This suggests that TET1 may be the potential reason for down-regulation of 5hmC in ALD.

The 5hmC epigenetic modification has already been linked to the progression of cancers and other diseases (19–21). Although it is largely unknown how 5hmC is involved in hepatocyte apoptosis in ALD, knockdown of TET1 has been shown to promote neuronal cell apoptosis by altering DNA methylation through the reduction of 5hmC (22). Interestingly, chronic alcohol consumption inhibits 5hmC levels in the livers of ALD human patients (16, 23). In addition, chronic alcohol consumption promotes hepatocyte apoptosis in a rat ALD model (24). Given the facts that knockdown of TET1 increases cell apoptosis, that chronic alcohol drinking promotes hepatocyte apoptosis, and that 5hmC is decreased in ALD patients, it is highly possible that this all promotes hepatocyte apoptosis by down-regulating TET1 expression and by suppressing 5hmC epigenetic changes, resulting in disrupted apoptotic signaling pathways. The current study was designed to clarify the molecular mechanisms related to how alcohol-mediated 5hmC is involved in hepatocyte apoptosis.

MATERIALS AND METHODS

Cell lines and reagents

Human OUMS29 hepatocyte-like cells were cultured as previously described (24, 25). A TUNEL staining kit was purchased from Roche (Basel, Switzerland)/MilliporeSigma (Burlington, MA, USA). Bcl-XL, HIF-1α, γ-H2aX, and cleaved poly(ADP-ribose) polymerase (PARP) used for immunoblotting experiments were from Cell Signaling Technology (Danvers, MA, USA). Iron chelator deferoxamine mesylate salt (DFO), 5-azacytidine (5-Aza), Bcl2, GAPDH, and α-tubulin antibodies used in immunoblotting were from MilliporeSigma. The 5hmC antibody was from Active Motif (Carlsbad, CA, USA). The TET1 mAb (GT1462) and TET1 pAb (PA5-72805) for Western blot were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The short hairpin RNA (shRNA)-TET1 plasmid was from GE Dharmacon (Lafayette, CO, USA).

Human subjects

The normal health liver samples were from the normal liver tissues adjacent to hepatic hemangioma. The normal health liver samples were from the Beijing 302 Hospital. The 13 human ALD tissue samples were from the Beijing 302 Hospital. Detailed information of patients is listed in Supplemental Table 2. All studies were approved by the Institutional Review Board of Beijing 302 Hospital.

Animal experimentation

Eight-week-old Long Evans female rats were purchased from Harlan Laboratories (South Easton, MA, USA). The ALD model was established as previously described. Basically, Long Evans rats were acclimated for 1 wk. Then rats were fed with an isocaloric diet or a liquid diet containing alcohol (37% of total calorie) for 8 wk (26). Eight and 9 rats were used in the control and alcohol groups, respectively. Rats were humanely killed, and livers and blood were collected for further analysis. The study protocol was approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital.

Immunoblotting

Total protein was extracted from animal tissues or cell lines treated as indicated using RIPA buffer. Protein concentrations were determined using the bicinchoninic acid assay. Fifty micrograms of protein was resolved in 10% SDS-PAGE gel and transferred to PVDF membrane. The proteins were detected by hybridizing with the indicated first antibody, followed by hybridization of the secondary antibody conjugated with horseradish peroxidase, visualized by Pierce ECL Western Blotting Substrate (Pierce, Rockford, IL, USA), and photographed with the Fujifilm Image System (Fuji, Tokyo, Japan). The dilutions of first antibodies were 1:1000 for Bcl-XL, 1:500 for HIF-1α, 1:1000 for γ-H2aX, 1:1000 for cleaved PARP, 1:500 for Bcl2, 1:3000 for GAPDH, and 1:500 for α-tubulin. The dilutions of secondary antibodies were 1:3000 mouse IgG-conjugated with horseradish peroxidase (HRP) and 1:10,000 for rabbit IgG conjugated with HRP. The membrane was incubated with Pierce ECL Plus Western Blotting Substrate, and images were taken using the Bio-Rad Gel Image System following the instruction manual (Bio-Rad, Hercules, CA, USA).

Aspartate aminotransferase and alanine transaminase assays

Rat blood samples were collected via cardiac puncture and placed in 1.5 ml Eppendorf tubes. Serum was separated from the samples by centrifuging at 3000 rpm for 30 min. Fifty microliters of serum samples was used to perform aspartate aminotransferase (AST) assay using the AST Activity Colorimetric Assay Kit (BioVision, Milpitas, CA, USA) or alanine aminotransferase (ALT) assay with the ALT Colorimetric Activity Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA) following the instruction manuals.

Real-time quantitative PCR

Tissue and cell samples were lysed with Trizol reagent (Thermo Fisher Scientific). RNA was extracted from lysed samples and quantified. One microgram of RNA was used to reverse transcribe to a cDNA using an iScript cDNA Synthesis Kit (Bio-Rad). cDNA was diluted with 180 ml distilled H2O. Two microliters of cDNA in combination with specific primers was used for real-time quantitative PCR reaction. Relative mRNA expression was calculated by comparison to internal control (GAPDH) using the ΔΔCt method. The primers used in the current study are listed in Supplemental Table 1.

Immunohistochemistry

For immunohistochemical assay, tissue slides were deparaffined with xylene and rehydrated with a serial dilution of ethanol. Rehydrated tissue slides were processed with antigen retrieval with antigen unmasking solution purchased from Vector Laboratories (Burlingame, CA, USA). The processed slides were blocked with 3% H2O2 prepared in methanol for 15 min to inhibit endogenous peroxidase; the slides were then blocked with 5% milk for 1 h to suppress nonspecific binding. Tissue slides were incubated with the first antibody overnight at 4°C and then incubated with the specific secondary antibody conjugated with biotin for 1 h at room temperature. The dilution of 5hmC antibody (Active Motif) was 1:4000. The dilution of secondary antibody was 1:100. The VectaStain Elite ABC HRP Kit (Vector Laboratories) was used to enhance protein signals according to the manufacturer’s instructions. The protein signal was visualized using the DAB Peroxidase (HRP) Substrate Kit (Vector Laboratories). Images were taken using the Nikon Imaging System (Nikon, Tokyo, Japan).

TUNEL assay

TUNEL assay was performed using the In Situ Cell Death Detection Kit, POD (MilliporeSigma). In short, 5 mm liver slides were deparaffined and rehydrated. The slides were treated with proteinase K working solution for 30 min and then processed according to the manufacturer’s instructions. Images were taken as previously described.

Blood alcohol concentration assay

Blood was taken using cardiac puncture and collected in heparin tubes. Blood samples (10–15 μl) were centrifuged at 1200 g for 5 min at 4°C. The resulting supernatants were then transferred to fresh Eppendorf tubes, and blood alcohol concentrations were measured using the Analox GM7 Micro-Stat (Analox Instruments, Lunenberg, MA, USA). All samples were run in duplicate.

Statistical analysis

All statistical analyses were performed by a Student t test comparing experimental groups to the control, except that the ANOVA analysis was used in certain experiments for group statistical analysis. A value of P < 0.05 was considered significantly different.

RESULTS

Chronic alcohol consumption elicits alcoholic liver disease

To investigate how chronic alcohol drinking affects 5hmC formation, after 8 wk of alcohol feeding, experimental rats were humanely killed and blood alcohol content determined. As expected and shown in Fig. 1A, alcohol concentration was significantly increased in ethanol-fed rats. Consistently, ethanol feeding suppressed body weights without significant effect on liver weight (Fig. 1B, C). In addition, it also resulted in the elevation of alanine aminotransferase (ALT) activity but not AST (Fig. 1D). To confirm ALD, hematoxylin and eosin staining was performed to evaluate the extent of fat in the livers. Fig. 1E shows that 8 wk of alcohol feeding induced fatty liver (Fig. 1E) and a significant increase in hepatocyte apoptosis (Fig. 1F).

Figure 1 .

Figure 1

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Chronic alcohol feeding resulted in hepatocyte apoptosis. A) Blood alcohol levels were determined in isocaloric liquid diet (Ctrl) and experimental liquid diet containing ethanol (37% of calories); n = 8 in control group and n = 9 in ethanol group. B, C) Body (B) and liver (C) weights of experimental rats were measured; n = 8 in control group and n = 9 in ethanol group. D) ALT and AST activities were measured in control and ethanol groups, n = 7. E) Representative hepatic hematoxylin and eosin (H&E)–stained images were obtained from control and ethanol groups. Scale bars, 25 µm. F) Representative TUNEL staining images were from control and ethanol groups. Top: low-power field; bottom: high-power field. Scale bars, 50 µm. Graph below images is quantitation data. Brown nuclear color staining indicates positive apoptosis signal; arrowhead points to cells with positive signals; n = 8 in control group and n = 9 in ethanol group. N.S., no significant difference. *P < 0.05, ***P < 0.001.

ALD is associated with decreased 5hmC formation

Epigenetic changes and hepatocyte apoptosis have been shown to be major features in the development of ALD (7, 24). 5hmC is a recently recognized epigenetic modification shown to participate in the progression of hepatocellular carcinoma and nonalcoholic liver diseases (16, 17). To investigate whether 5hmC is involved in ALD, 5hmC levels were determined in rats and human ALD liver samples by dot-blot analysis and immunohistochemistry. The results suggested that 5hmC is down-regulated in the rat model and in human samples compared to the relative controls (Fig. 2A, B). The TET family enzymes were analyzed in order to clarify if TET family enzymes down-regulated 5hmC. Consistent with a previous report (16), TET2 and TET3 expression levels were not significantly different between control and ALD samples (Fig. 2C and Supplemental Fig. 1). By analyzing TET1, TET2, and TET3 mRNA expression in rat and human livers using rat liver samples and data from the GTEx project, it was found by real-time quantitative PCR that TET1 mRNA was barely detectable in rat liver samples (ΔCt is about 15, CtTET1 − CtGAPDH, n = 5). Further analysis of data obtained by RNA sequencing from the GTEx project (https://www.gtexportal.org/home/) revealed that TET1 mRNA is expressed in human livers, but its expression is relatively low compared to TET2 and TET3 (Fig. 2D, n = 119). Because TET2 and TET3 were not altered in ALD, the current findings could not explain the down-regulated 5hmC in ALD (Fig. 2C), so it was suggested that TET1 may be involved. Before TET1 protein expression was determined in rat livers, the TET1 antibody used in this study was carefully evaluated. We used liver protein lysates derived from wild-type and TET1 knockout mice (27) as positive and negative controls. The protein lysates of OUMS29 cells transduced with shRNA-luciferase (shLuc) were also used to validate the TET1 antibody. The TET1 MW is about 220 kDa in mouse embryonic stem cells (27), and so is the TET1 antibody used in this study. However, human TET1 is larger than 250 kDa according to the TET1 antibody data sheets. Mouse TET1 protein (below 250 kDa) was detected in wild-type mice but not in TET1-knockout mice. In addition, human TET1 was observed in shLuc-OUMS29 cells (Supplemental Fig. 2), suggesting that the used TET1 antibody is valid. We then determined TET1 protein and found it was moderately expressed in rat livers (Fig. 2E) (16, 17).

Figure 2 .

Figure 2

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Chronic alcohol drinking suppressed 5hmC expression independent of TET2 and TET3. A) Left: global 5hmC levels were determined in control (Ctrl) and ethanol rat livers by using 5hmC ELISA kit. Right: representative immunohistochemical images of 5hmC and quantification data were derived from control and ethanol rat livers. Scale bars, 25 µm; n = 8 in control group and n = 9 in ethanol group. B) Representative immunohistochemical images of 5hmc and quantification data were obtained from normal healthy donors’ livers (NHL) and patients with alcoholic liver diseases (ALD), n = 4 in NHL and 13 in ALD. Top scale bars, 100 µm. C) TET2 and TET3 mRNA were measured in control- and ethanol-group rat livers; n = 8 in control group and n = 9 in ethanol group. D) TET1, TET2, and TET3 were analyzed in normal rat livers (n = 5) as well as NHL (n = 119 from GTEx). E) TET1 protein was determined in 5 rat liver samples (left) by immunoblotting. *P < 0.05, ***P < 0.001.

TET1 expression is decreased and associated with apoptosis in ALD

TET1 expression was measured in rat ALD samples and was shown to be reduced in ALD tissues compared to normal controls (Fig. 3A and Supplemental Fig. 1). The antiapoptotic proteins Bcl2 and Bcl-XL were determined in control and ALD rat samples. Consistently, elevated apoptotic activities were observed in ALD samples as antiapoptotic proteins were down-regulated (Fig. 3B). To investigate if the decreased TET1 expression may be one of the underlying mechanisms causing hepatocyte apoptosis, we inhibited TET1 expression using shRNA-TET1. This strategy successfully suppressed TET1 mRNA and protein expression, as well as its functional activity in catalyzing 5hmC formation (Fig. 3C). After validating the depletion of TET1, hepatocyte-like cell apoptosis was examined by flow cytometry with propidium iodide/annexin V stain and immunoblotting analysis. Intriguingly, TET1 deficiency significantly increased early and late apoptotic/necrotic cells (Fig. 3D). TET1 deficiency also reduced viable cells (Fig. 3D). The TET1 deficiency–induced apoptosis was further validated by the knockdown of TET1, which promoted the robust elevation of cleaved PARP expression (Fig. 3E). Clearly, down-regulation of TET1 instigates hepatocyte apoptosis.

Figure 3 .

Figure 3

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Down-regulated TET1 in ALD was associated with apoptosis. A) TET1 and α-tubulin expression levels were determined in control (Ctrl) and ethanol rat livers; shown at right are quantification data. B) Bcl-2, Bcl-XL, and α-tubulin levels were measured in rat ALD samples by immunoblotting. Shown at right are quantitation data. C) 5hmC was determined using dot-blot protocols. TET1 and α-tubulin were analyzed using immunoblotting. D) Early apoptotic cells, late apoptotic cells, and viable cells were determined in shLuc- and shTET1-treated OUMS29 cells by flow cytometry. E) Cleaved PARP and α-tubulin were analyzed in shLuc- and shTET1-treated OUMS29. *P < 0.05, **P < 0.01, ***P < 0.001.

Down-regulated TET1 increases proapoptotic gene Harakiri-BCL2 interacting protein expression

To determine how alcohol-associated TET1 down-regulation affects hepatocyte apoptosis, we evaluated gene expression of antiapoptotic proteins and proapoptotic proteins in shRNA-TET1–treated human hepatocyte–like cells with detectable TET1 mRNA expression as determined by real-time quantitative PCR (Fig. 4A). While knockdown of TET1 significantly suppressed its expression in hepatocyte-like cells, this did not alter mRNA expression of any of the antiapoptotic proteins studied, including B-cell lymphoma 2 protein (Bcl2), Bcl-2–like protein 2 (Bcl-w), and B-cell lymphoma–extra large protein (Bcl-XL) (Fig. 4B). We then examined several proapoptotic genes, including Bcl-2-like protein 11 (BIM), BCL2 antagonist/killer 1 (BAK), bcl-2-like protein 4 (BAX), BH3 interacting-domain death agonist (BID), Harakiri-BCL2 interacting protein (HRK), and BCL2 interacting killer (BIK). We observed that HRK and BIK are seemingly increased in human hepatocyte–like cells with TET1 knocked down (Fig. 4C). To determine if the observed effect is reproducible in an in vivo rat ALD model, HRK and BIK expression was measured in liver samples derived from control-fed and ALD rats. Interestingly, only up-regulated HRK was found in ALD samples (Fig. 4D). Therefore, ALD-associated TET1 decrease may promote hepatocyte apoptosis by enhancing HRK expression.

Figure 4 .

Figure 4

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Proapoptotic gene HRK was elevated in human hepatocytes treated with shTET1. A–C) TET1 (A), antiapoptotic genes (Bcl2, Bcl-w, and Bcl-XL) (B), and proapoptotic genes (Bim, Bak, Bax, Bid, HRK, and BIK) (C) were analyzed in human hepatocytes treated with shLuc or shTET1. D) HRK and BIK were determined in rat liver samples derived from control (Ctrl) and ethanol groups. *P < 0.05.

Decreased TET1 enhances DNA damage and GADD45α expression

Alcohol drinking is associated with DNA damage in human cells (28). The current study identified that down-regulated 5hmC and TET1 are associated with hepatocyte apoptosis, one of the downstream events observed in DNA damage responses. It has also been suggested that mutant IDH1R132H alters DNA repair and sensitivity to DNA damage (29). Because one of the mechanisms by which mutant IDH1R132H promotes carcinogenesis is suppression of TET1 enzymatic activity (30), it is possible that down-regulated TET1 may likewise elicit DNA damage in hepatocytes in ALD. Indeed, knockdown of TET1 significantly induced the expression of γ-H2aX (Fig. 5A), a DNA damage biomarker (31). Similarly, γ-H2aX expression was found to be higher in ethanol-fed rat livers than in control-fed ones (Fig. 5B). γ-H2aX is a phosphorylated form of histone H2aX and this phosphorylation is catalyzed by 3 different kinases, including ataxia telangiectasia and Rad3-related protein (ATR), DNA-dependent protein kinase catalytic subunit (PRKDC), and ataxia–telangiectasia–mutated protein (ATM). We therefore analyzed the gene expression of ATM, ATR, and PRKDC in human hepatocyte–like cells treated with shRNA-TET1. Interestingly, knockdown of TET1 did not alter the expression of those genes (Fig. 5C). Because the MRE11/RAD50/NBS1 (MRN) complex is the major component phosphorylating ATM leading to H2aX phosphorylation (32), it is reasonable to determine if the gene expression levels of MRN complex are affected by TET1 down-regulation. However, knockdown of TET1 did not alter the expression of MRE11, RAD50, or NBS1 (Fig. 5D). The p53 binding protein 1 (53BP1), another important mediator involved in DNA repair, was also examined, but its expression was not affected (Fig. 5E).

Figure 5 .

Figure 5

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Down-regulation of TET1 resulted in elevated GADD45α expression and DNA damage. A) TET1, γ-H2ax, and α-tubulin were determined in shLuc- or shTET1-treated OUMS29 cells. B) γ-H2ax and α-tubulin were analyzed in rat livers of control (Ctrl) and ethanol groups. Right: quantification data. C) mRNA expression of protein kinases for H2aX, including ATR, PRKDC, and ATM, was determined in shLuc- or shTET1-treated OUMS29 cells. D, E) mRNA expression of MRN complex components, including MRE11, NBS1, and RAD50 (D) as well as TP53BP1 (E), was analyzed in OUSM29 treated as indicated. F) Genotoxic stress-responsive gene GADD45α was measured in OUSM29 cells treated with shLuc or shTET1 and in rat liver samples derived from control or ethanol groups. *P < 0.05.

Posttranslational modification of proteins, including ubiquitination, is another important component of DNA repair mechanisms (33), and we analyzed expression of genes associated with this process for further clarification of how alcohol-associated TET1 down-regulation may stimulate DNA damage. Results suggest that the ubiquitin conjugating enzyme E2 N (UBC13) and the ring finger protein 8 (RNF8) are not influenced. (Supplemental Fig. 3). Several other DNA repair–associated proteins were then investigated (34). Neither RAD51, RAD54, BRCA1, RPA1, XRCC2, XRCC3, XRCC5, nor XRCC6 was found to be altered (Supplemental Fig. 4). Recently, the TET1-mediated 5hmC formation has been identified as a mechanism potentially responsible for active DNA demethylation (35). The current study indicates that alcohol consumption is associated with decreased 5hmC, which should therefore theoretically lead to DNA hypermethylation. But DNA hypomethylation has been reproducibly identified in ALD and hepatocellular carcinoma patients (28), suggesting that compensatory mechanisms are responsible. However, TET2 and TET3 were found unchanged between normal controls and ALD samples in both a rat ALD model (Fig. 2E) and in human tissue (16). Clearly, other reasons are responsible for DNA hypomethylation. GADD45α, a marker of DNA damage, is another potential mechanism involved in this repair-mediated DNA demethylation (36). Intriguingly, the expression levels of GADD45α were found elevated in both shRNA-TET1–challenged human hepatocyte–like cells and ethanol-fed rat livers (Fig. 5F), suggesting that up-regulated GADD45α may be induced by the hypomethylation.

Removing cosubstrate of TET1 by desferrioxamine promotes DNA damage and apoptosis

To determine if a biochemical approach by inhibiting TET1 activity could result in DNA damage and apoptosis in hepatocytes, we targeted TET1 in OUMS29 cells by depleting its cosubstrate using DFO, an iron chelator (Fig. 6A). Treating hepatocyte-like cells with 50 and 100 µM DFO substantially suppressed 5hmC production at d 1, 2, and 3, respectively (Fig. 6B). Targeting TET1 with DFO also significantly enhanced HRK and GADD45α expression (Fig. 6C, D). More importantly, DFO treatments robustly induced apoptosis and DNA damage (Fig. 6E and Supplemental Fig. 5). The results clearly demonstrated that mimicking ALD-associated down-regulation of TET1 with molecular (Figs. 4 and 5) and biochemical (Fig. 6) approaches can result in elevation of HRK and GADD45α, in turn leading to increased DNA damage and apoptosis.

Figure 6 .

Figure 6

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Inhibiting TET1 activity with iron chelator DFO led to increased apoptosis and DNA damage in hepatocytes. A) Cartoon illustrating TET1 enzymatic reaction. B–D) 5hmC levels (B), HRK (C), and GADD45α (D) mRNA expression were analyzed in human hepatocyte–like cells treated with 0, 50, and 100 µM DFO for 1, 2, and 3 d. E) Cleaved PARP (apoptosis marker), γ-H2aX (DNA damage marker), and GAPDH were determined in human hepatocyte–like cells treated as indicated. **P < 0.01, ***P < 0.001.

5-Aza treatment mimics effects of TET1 down-regulation on apoptosis and DNA damage

The TET1 mediates gene expression via modifying methylation either in the gene body or promoter region (37, 38). As TET1 is down-regulated in human and rat ALD samples, it is possible that the up-regulation of HRK and GADD45α observed in ALD is affected by TET1-mediated DNA methylation. To clarify this, a DNA methylation inhibitor, 5-Aza, was used to challenge human hepatocyte–like cells treated with shRNA-TET1. Consistently, knockdown of TET1 substantially promoted HRK and GADD45α expression in human hepatocyte–like cells (Fig. 7A, B). Challenging shRNA-TET1–treated hepatocytes with 5-Aza further raised HRK and GADD45α expression (Fig. 7A, B). In this circumstance, 5-Aza treatment could likewise amplify the effects of shRNA-TET1 on apoptosis and DNA damage. This is indicated by more greatly elevated expression of cleaved PARP and γ-H2aX (Fig. 7C and Supplemental Fig. 5). This implies that ALD-associated down-regulation of TET1, leading to altered DNA methylation of genes, increases HRK expression and consequently greater DNA damage and apoptosis.

Figure 7 .

Figure 7

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Repressing DNA methylation by using 5-Aza promoted HRK and GADD45α expression as well as apoptosis and DNA damage. A, B) HRK (A) and GADD45α (B) expression was analyzed in shLuc- or shTET1-OUMS29 cells treated with vehicle control or 10 µM 5-Aza for 0, 1, 2, and 3 d. C) Immunoblotting results of TET1, cleaved PARP, γ-H2aX, and α-tubulin were obtained from OUMS29 cells treated as indicated. *P < 0.05, ***P < 0.001.

DISCUSSION

The current study identified that chronic alcohol feeding promotes hepatocyte apoptosis, TET1 down-regulation, and 5hmC reduction in a rat ALD model. Decreased 5hmC levels were also observed in human ALD samples. Using molecular and biochemical approaches to target TET1, it was demonstrated that inhibiting TET1 activity substantially enhances apoptosis and DNA damage as well as promotes HRK and GADD45α expression in hepatocytes. Interestingly, the treatment of the DNA methylation inhibitor 5-Aza was found to mimic the effects of TET1 inhibition on hepatocytes, suggesting that TET1 may modulate apoptosis and DNA damage in hepatocytes via DNA methylation.

Recently, 5hmC has been suggested to be down-regulated in ALD patients compared to normal healthy liver controls (16, 23). In both studies, 5hmC was found decreased, but not to any significant extent. The current study investigated 5hmC levels in a rat ALD model as well as in human ALD samples and found that 5hmC is significantly decreased in both. In fact, a previous study showed that 5hmC is decreased in 3 animal models of liver damage, including bile duct ligation, carbon tetrachloride injection, and feeding with a methionine- and choline-deficient diet (16). This 5hmC phenotype was reproducibly observed in nonalcoholic fatty liver disease (39). Although TET2 and TET3 were suggested to be involved in down-regulation of 5hmC in liver fibrosis animal models, they were not significantly altered in human ALD samples (16). This hinted at the possible involvement of TET1 in ALD. However, TET1 mRNA was not detected by RT-PCR in an experimental rat model (16), though any conclusion may be qualified by the limitation of the technique. While the current study did detect TET1 mRNA in rat liver samples, it was found to be extremely low. Interestingly, TET1 mRNA is also very low in human livers, but TET1 mRNA levels are comparable in other organs (GTEx data) such as the brain. Nevertheless, a recent study has indicated that brain TET1 protein is involved in cocaine action (37). The actual TET1 protein was found moderately expressed in mouse and human liver samples, however. In this context, the TET1 protein expression level may be a crucial determinant in 5hmC changes in liver damage animal models. Indeed, the current study found that TET1 is down-regulated in rat and human ALD samples.

Hepatocyte apoptosis is an important driving force of the progression of ALD. Although studies have suggested putative molecular mechanisms to be involved in hepatocyte apoptosis (24, 40), it is unclear if the newly identified 5hmC epigenetic modification affects hepatocyte apoptosis leading to ALD progression. The current study shows that down-regulation of TET1 decreases 5hmC in rat and human ALD. More importantly, the reduced TET1 expression also induced apoptosis. Additionally, down-regulation of TET1 also caused DNA damage. Further studies disclosed that HRK and GADD45α are elevated in human hepatocytes treated with TET1 knockdown and in a rat ALD model, signaling the possible involvement of the TET1/HRK/GADD45α signaling pathway in disease progression. As down-regulation of TET1 results in reduced 5hmC, which in turn leads to diminished DNA demethylation, knockdown of TET1 theoretically would suppress HRK and GADD45α expression due to DNA hypermethylation. This finding seems to contradict the concept that TET1-mediated 5hmC promotes DNA demethylation. However, TET1 was demonstrated as having the dual function of activating or suppressing transcriptional regulation. However, there were more activated genes than repressed genes found in TET1 knockdown cells (38), indicating that alcohol-associated down-regulation of TET1 may promote gene expression through inhibition of DNA methylation.

DNA methylation is a critical epigenetic modification controlling gene expression. As down-regulated TET1 enhances gene expression by suppression of DNA methylation, it is likely that elevated HRK and GADD45α in ALD and human hepatocytes treated with TET1 knockdown may be regulated through this mechanism. By searching available databases (37, 38), we found that HRK is up-regulated in shRNA-TET1 (shTET1)-treated neuron cells. Interestingly, knockdown of TET1 promoted 5hmC formation in the body of the HRK gene, suggesting that TET1 may inhibit HRK expression via methylation. The similar observation that knockdown of TET1 promotes gene expression was found with GADD45α (38). This further strengthens the hypothesis that down-regulation of TET1 in ALD promotes HRK and GADD45α expression by inhibition of DNA methylation. One might expect that 5-Aza treatment would replicate the effects of TET1 down-regulation on HRK and GADD45α as well as any functional outcomes. Indeed, inhibiting DNA methylation with 5-Aza treatment did raise HRK and GADD45α expression, thus leading to increased apoptosis and DNA damage. These data, taken together, imply that decreased TET1 in ALD promotes apoptosis and DNA damage by modifying DNA methylation.

Even though TET1 was down-regulated in human and rat ALD, knockdown of TET1 did not completely mimic gene changes observed in ALD. In addition, down-regulated 5hmC should result in DNA hypermethylation, but global DNA hypomethylation has been found in experimental animals (9, 41), suggesting that several directions will need to be further investigated to clarify the role of 5hmC in ALD progression. As GADD45α is another enzyme potentially involved in DNA demethylation, and down-regulation of TET1 in ALD raises its expression, GADD45α may be one of the compensatory mechanisms responsible for DNA hypomethylation in ALD. Additionally, it would be interesting to investigate a role of TET2 and TET3 in ALD progression, even though their expression levels were not altered significantly in rat and human ALD, given the reason that they are relatively abundant TET family proteins in the liver. Nevertheless, our data demonstrated that down-regulated TET1 in ALD results in a decrease in 5hmC formation. Rather than suppressing gene expression, this down-regulated TET1 promotes HRK and GADD45α expression by inhibition of DNA methylation. Elevated HRK and GADD45α may affect apoptosis and DNA damage, thus partly explaining how TET1 and 5hmC are involved in ALD. Further investigations regarding either overexpressing TET1 or knockout of TET1 in hepatocytes may clarify the molecular mechanisms by which TET1 influences disease progression. Through the use of cosubstrates of TET1, including iron and 2-oxoglutarate, to treat ALD animal models, we may obtain information that will permit us to determine whether targeting TET1 may potentially be a therapy for ALD.

Supplementary Material

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

Click here for additional data file. (2.3MB, doc)

ACKNOWLEDGMENTS

The authors thank Z. Cheng (Warren Alpert Medical School of Brown University and Rhode Island Hospital), X. Bai (Warren Alpert Medical School of Brown University and Rhode Island Hospital), and G. Zhou (Beijing 302 Hospital) for their technical support. The GTEx Project is supported by the Common Fund of the Office of the Director of the U.S. National Institutes of Health (NIH), and by the National Cancer Institute, National Human Genome Research Institute, National Heart, Lung, and Blood Institute, National Institute on Drug Abuse, National Institute of Mental Health, and National Institute of Neurological Disorders and Stroke. The data used for the analyses described here were obtained from the GTEx data set. Funding for this work was provided by NIH (P20GM103430-12), the Rhode Island Foundation (134279), American Association for the Study of Liver Diseases pinnacle research award, the 302 Hospital President Foundation (YNKT 2014018), and Brown University Karen T. Romer Undergraduate Teaching and Research awards. The authors declare no conflicts of interest.

Glossary

5-Aza

5-azacytidine

ALD

alcoholic liver disease

ALT

alanine aminotransferase

AST

aspartate aminotransferase

ATM

ataxia–telangiectasia–mutated protein

ATR

ataxia telangiectasia and Rad3 related protein

BIK

BCL2 interacting killer

DFO

deferoxamine mesylate salt

GTEx

Genotype-Tissue Expression

5hmc

5-hydroxymethylcytosine

HRK

Harakiri-BCL2 interacting protein

HRP

horseradish peroxidase

MRN

MRE11/RAD50/NBS1

PARP

poly(ADP-ribose) polymerase

PRKDC

DNA-dependent protein kinase catalytic subunit

shLuc

shRNA-luciferase

shRNA

short hairpin RNA

shTET1

shRNA-TET1

TET1

methylcytosine dioxygenase TET1

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

AUTHOR CONTRIBUTIONS

C. Ji, K. Nagaoka, J. Zou, and C.-K. Huang were responsible for the study concept and design; C. Ji, K. Nagaoka, J. Zou, S. Casulli, K. Y. Cao, H. Zhang, Y. Iwagami, R. I. Carlson, K. Brooks, J. Lawrence, W. Mueller, and C.-K. Huang acquired data; C. Ji, K. Nagaoka, J. Zou, and C.-K. Huang analysed and interpreted the data; C.-K. Huang drafted the manuscript; R. I. Carlson and J. R. Wands revised the manuscript; S. Casulli, Y. Iwagami, and C.-K. Huang provided technical support; C. Ji and S. Lu provided material support; C. Ji, J. R. Wands, and C.-K. Huang obtained funding; C.-K. Huang supervised the study; and all authors approved the final manuscript.

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