Rdh13 deficiency weakens carbon tetrachloride-induced liver injury by regulating Spot14 and Cyp2e1 expression levels

Abstract

Mitochondrion-localized retinol dehydrogenase 13 (Rdh13) is a short-chain dehydrogenase/reductase involved in vitamin A metabolism in both humans and mice. We previously generated Rdh13 knockout mice and showed that Rdh13 deficiency causes severe acute retinal light damage. In this study, considering that Rdh13 is highly expressed in mouse liver, we further evaluated the potential effect of Rdh13 on liver injury induced by carbon tetrachloride (CCl₄). Although Rdh13 deficiency showed no significant effect on liver histology and physiological functions under regular culture, the Rdh13^−/− mice displayed an attenuated response to CCl₄-induced liver injury. Their livers also exhibited less histological changes and contained lower levels of liver-related metabolism enzymes compared with the livers of wild-type (WT) mice. Furthermore, the Rdh13^−/− mice had Rdh13 deficiency and thus their liver cells were protected from apoptosis, and the quantity of their proliferative cells became lower than that in WT after CCl₄ exposure. The ablation of Rdh13 gene decreased the expression levels of thyroid hormone-inducible nuclear protein 14 (Spot14) and cytochrome P450 (Cyp2e1) in the liver, especially after CCl₄ treatment for 48 h. These data suggested that the alleviated liver damage induced by CCl₄ in Rdh13^−/− mice was caused by Cyp2e1 enzymes, which promoted reductive CCl₄ metabolism by altering the status of thyroxine metabolism. This result further implicated Rdh13 as a potential drug target in preventing chemically induced liver injury.

Keywords

retinol dehydrogenase 13 / carbon tetrachloride / acute liver injury / Cyp2e1 / Spot14

Introduction

Mitochondrion-localized retinol dehydrogenase 13 (Rdh13) is a member of short-chain dehydrogenase/reductase (SDR) participating in vitamin A metabolism in both humans and mice [¹,²]. SDR belongs to a large family of nicotinamide adenine dinucleotide/triphosphopyridine nucleotide phosphate (NAD(P)H)-dependent oxidoreductase [³]. The members of SDR share the same cofactors and substrates, although they exist in different organelles, such as the mitochondria, peroxisomes, and endoplasmic reticulum [⁴,⁵]. As a member of the SDR superfamily, Rdh13 displays high sequence similarity to Rdh11, Rdh12, and Rdh14 and belongs to short-chain-retinol dehydrogenases with dual-substrate specificity (RDH family) [⁶–¹⁰]. The concentrate on the inner mitochondrial membrane of Rdh13 suggests that it plays different roles in contrast to Rdh11, Rdh12, and Rdh14 localized in the endoplasmic reticulum [¹¹]. SDRs play significant roles in the metabolism of lipid, protein, carbohydrate, and chemical compounds. In the human genome database, at least 63 gene members have been identified, but the cellular functions of most oxidoreductases, including Rdh13, are not fully understood [⁴,⁵]. Previously, we generated an Rdh13 knockout mouse model and found the important roles of Rdh13 in protecting the retina from light damage; this finding was also observed in Rdh11 and Rdh12 mutant mice [⁶–¹⁰]. Furthermore, Rdh13 is highly expressed in mouse liver such that its amount in the liver is 3–10 times greater than that observed in other tissues, suggesting that the Rdh13 will play significant roles in liver metabolism. The liver can renew by itself after being attacked by toxic substances, hepatitis virus, and surgery, and liver parenchymal cells in G₀ phase initiate proliferation upon these stimuli [¹²–¹⁵]. Both hepatocytes and hepatic stellate cells are indispensable in retinoid metabolism [¹⁶]. Numerous chemical compounds, such carbon tetrachloride (CCl₄), are normally used to generate animal models in studying the progress and mechanism of liver injury and regeneration [¹²,¹³,¹⁶]. Free trichloromethyl radical, the metabolites of CCl₄ catalyzed by Cyp2e1, can bind to macromolecules, such as proteins, nucleic acids, and lipids, to prevent them from eliciting functions that damages the liver [¹⁷]. Acute liver damage caused by CCl₄ is full of inflammation, apoptosis, hepatocyte necrosis, and then is completely restored by liver regenered [¹⁸,¹⁹]. The progress and mechanism of acute liver damage are relatively well characterized for histological, biochemical, and molecular alteration [²⁰]. Substantial evidence reveal that vitamin A and its metabolites, such as retinoic acid, are involved in liver injury, regeneration, fibrosis, and tumorigenesis [²¹,²²].

In the present study, we evaluate the potential effect of Rdh13 in liver metabolism, especially during CCl₄-induced liver injury and regeneration by utilizing Rdh13^−/− mouse model. No remarkable difference is observed between wild-type (WT) and Rdh13^−/− mice with respect to liver enzymes and liver histology. However, after CCl₄ treatment, the Rdh13^−/− mice display attenuated liver injury in contrast to the WT controls. Rdh13 deficiency protects liver cells from apoptosis. Furthermore, proliferative cells decrease after CCl₄ exposure. Rdh13 deficiency decreases the expression levels of Spot14 and cytochrome P450 (Cyp2e1). This result suggests that alleviated liver damage induced by CCl₄ in Rdh13^−/− mice is due to Cyp2e1 enzyme, which promotes reductive CCl₄ metabolism by changing status of thyroxine metabolism, further implicating Rdh13 as a potential drug target in preventing chemically induced liver injury.

Materials and methods

Animals

Rdh13^−/− mice were obtained as previously reported [⁹]. WT and Rdh13^−/− mice (8–10 weeks old) with mixed C57BL/6 and 129/Sv background were used in the study. The food and water intake were provided freely, and the mice were raised under specific pathogen-free conditions at a constant room temperature of 22–24 °C at 12 h light/dark cycle. All animal experiments were authorized by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.

CCl₄ induced acute liver injury

CCl₄ was used to induce acute liver injury. WT and Rdh13^−/− mice with the same ratio of male and female were intraperitoneally injected with a single dosage of CCl₄ (1.0 mL/kg body weight of 20% CCl₄ diluted in olive oil) [⁴,⁵,¹¹]. Four mice in each cohort were killed at 0, 3, 6, 12, 24, 48, and 72 h after CCl₄ treatment. Two hours before sacrificing, single dosage of bromodeoxyuridine (Brdu) was intraperitoneally injected at the dosage of 50 mg/kg mouse weight (0.2% ddH₂O solution).

Histological analysis

Formalin-fixed liver samples were processed overnight and then paraffin-embedded according to standard procedure. Liver sections were examined by hematoxylin and eosin (H&E) staining and subsequently analyzed under a light microscope (Nikon i90).

Serum biochemical analyses

The serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and other parameters were detected by the automatic biochemical analyzer according to the manufacturers’ instructions.

Terminal deoxynucleotidyl transferase-mediated uDP nick-end labeling (TUNEL) assay

We used the TUNEL assay (Cell Death Detection Kit, Promega) to assess apoptosis. Quiescent and DNase I pretreated WT livers were used as negative and positive controls, respectively. TUNEL-stained liver sections in each group at each time point after CCl₄ injection were examined as previously described [¹⁶]. The average percentage of apoptotic hepatocytes was compared between the two genotypes.

Immunohistochemistry

Immunohistochemistry analysis was performed by following the manufacturers’ instructions of VECTASTAIN®ABC system (Vector Laboratories). Mouse antiBrdu (Sigma), rabbit antiCyclin D1 (Cell Signaling Technology), and rabbit antiCaspase-3 (Abcam) were used. By counting Brdu labeled and total hepatocytes in 10 high-power fields (400×), Brdu incorporation of hepatocytes was calculated for each liver sample.

RNA isolation and quantitative real-time reverse transcription polymerase chain reaction (PCR)

Total RNA was prepared from mouse liver tissues using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. Quantitative PCR was carried out with SYBR Green real-time PCR Master Mix (Takara). The reactions were performed on an Eppendorf Mastercycler system, and the cycling parameters were reported previously [¹³]. Samples were run in triplicate, and the expression of specific transcript was normalized against b-actin. The primers used are listed in Supplementary Table S1.

Western blot analysis

Extraction and quantitative analysis of protein were performed as previously reported [⁹]. The primary antibody of Cyp2e1 (Sigma), caspase-3 (Abcam), Cyclin D1 (Cell Signaling Technology), Spot14 (Abcam), Bcl-2 (Abcam), and Bax (Abcam) were used in Western blot analysis, and glyceraldehyde 3-phosphate dehydrogenase (Cell Signaling Technology) serves as a loading control.

Statistical analysis

All arguments investigated are presented as mean±standard error of the mean (S.E.M.). Comparisons between the two groups were performed by two-tailed unpaired Student’s t-test. P values less than 0.05 were considered statistically significant.

Results

Rdh13 deficiency exhibited no marked effect on liver functions and histology

We performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) on major mouse tissues to examine the expression profile of Rdh13 in mice. We found that Rdh13 is widely expressed, and the expression levels vary considerably in different tissues. Meanwhile, the highest level was found in liver tissue, as shown in Supplementary Fig. 1, suggesting an essential functional compartment of Rdh13 in mice. Therefore, we first determined the biochemical parameters of liver functions in WT and Rdh13^−/− mice, which have been generated previously [⁹]. Unexpectedly, although Rdh13^−/− mice showed decreased level of liver function albeit ALT, no significant variation was observed in biochemical parameters including ALT, AST, and albumin (ALB) compared with WT mouse (Fig. 1A). In addition, liver histology displayed no discernable difference between Rdh13^−/− and WT mice (Fig. 1B). These data suggested an indispensable role of Rdh13 in liver biochemistry or histology of mice at basal condition.

Targeted deletion of Rdh13 alleviates CCl₄-induced hepatic injury

Given that Rdh13 deficiency showed no marked effect on basal liver biochemistry and histology, we decided to challenge the mutant mice with CCl₄ administration intraperitoneally because CCl₄-induced acute liver injury model is widely used to evaluate the progress and mechanism of liver injury and regeneration. As expected, all biochemical parameters tested, such as ALT, ALP, ALB, globulin, total cholesterol, AST, and triglyceride levels, respond to CCl₄ treatment to some degree. However, no significant difference was found between WT and Rdh13^−/− mice except for ALT (Fig. S2). Rdh13^−/− mice showed significantly lower levels of ALT after CCl₄ administration compared with WT mice. The serum ALT level in both genotypes peaked at 24 h and decreased at 48 h to 200 U/L in Rdh13^−/− mice, whereas serum ALT level was 1800 U/L in WT mice (Fig. 2A). Meanwhile, liver histology displayed marked vacuolization, necrosis, inflammation, and sinusoidal dilatations upon CCl₄ administration in both genotypes. However, these histological changes were milder in Rdh13^−/− mice than those observed in the WT controls (Fig. 2B). These data suggested that Rdh13 is involved in regulating the responses to CCl₄-induced acute liver injury.

Reduced hepatocyte apoptosis in Rdh13^−/− liver after CCl₄ challenge

The hepatic apoptosis and following cell proliferation are essential responses to CCl₄ treatment in mouse model. Therefore, we further assessed the hepatic apoptosis in WT and Rdh13^−/− mice upon CCl₄ treatment. We found that the amount of apoptotic hepatocytes decrease at 24, 48, and 72 h after CCl₄ exposure in Rdh13^−/− mice and lower than that in WT mice, as indicated by the results of TUNEL analysis (Fig. 3A and 3B). Caspase-3 is an evolutionary conserved cysteine protease and plays a central role in apoptotic cell death pathways [²³,²⁴]. We determined the expression level of caspase-3 by immunohistochemistry and Western blot. The results indicated that the amount of caspase-3 immune-reactive cells are significantly lesser in Rdh13^−/− mice compared with that in WT mice 24, 48, and 72 h after CCl₄ treatment (Fig. 3C). Consistent with this finding, caspase-3 protein levels in the liver showed a weak response to CCl₄ in the absence of Rdh13. Furthermore, antiapoptotic protein Bcl-2 increased and apoptotic protein Bax decreased in Rdh13^−/− livers relative to WT controls during CCl₄-induced liver injury (Fig. 3D). These results suggested that Rdh13 deficiency exhibited a protective effect against CCl₄-induced hepatocyte apoptosis.

Reduced compensatory response in hepatocellular proliferation post CCl₄-exposure in Rdh13^−/− mice

After CCl₄-induced injury, liver mass is rapidly replenished [¹²]. The effect of Rdh13 on liver regeneration after acute toxic liver injury was evaluated. Liver/body weight ratio reflects the liver proliferation status. However, no gross divergence was observed between WT and Rdh13^−/− mice (Fig. S3). Hepatocyte compensatory proliferation was further assessed by Brdu incorporation. Compared with WT mice, the Rdh13^−/− mice had lower degree of Brdu incorporation during liver regeneration. DNA synthesis peaked at 48 h post-CCl₄ exposure. More than 60% of the hepatocytes were Brdu positive in the WT liver, whereas less than 45% of the hepatocytes in Rdh13^−/− livers were Brdu positive (Fig. 4A and 4B). Cyclin D1 is a key molecule regulating the progress of G₁ phase in cell cycle [²⁵–²⁷]. We determined whether reduced cell proliferation is due to the altered expression of Cyclin D1 in the liver of Rdh13^−/− mice. The results showed that the expression levels of Cyclin D1 mRNA by qRT-PCR (Fig. 4C) and protein detected by either immunohistochemistry (Fig. 4D) or Western blot (Fig. 4E) were lower in Rdh13^−/− liver than those in WT controls after the CCl₄ treatment. Extracellular signal-regulated kinase (ERK) is also a significant factor for Cyclin D1 induction in mid-late G₁ phase [²⁸,²⁹]. Hepatocyte DNA replication and Cyclin D1 accumulation are mainly associated with ERK activation [³⁰]. Our result indicated that ERK activation in the absence of Rdh13 was reduced and lower compared with that in WT livers upon CCl₄ administration (Fig. 4F). All these data indicated the reduced compensatory response in hepatocellular proliferation post-CCl₄ exposure in Rdh13^−/− mice, at least in part, is due to the attenuated activation of ERK and reduced expression of Cyclin D1.

Expression levels of Cyp2e1 and Spot14 decreased in Rdh13^−/− livers after CCl₄ administration

CCl₄ toxicity is caused by the metabolite (trichloromethyl free radical) of CCl₄ by Cyp2e1 in the liver. We detected the expression levels of mRNA and protein of Cyp2e1 in both WT and Rdh13^−/− livers. As shown in Fig. 5A and 5B, the amounts of Cyp2e1 mRNA by qRT-PCR and protein detected by Western blot were reduced in Rdh13-deficient mice, especially at 48 h post-CCl₄ exposure, and lower compared with those in WT mice. Cyp2e1 regulation is particularly complex. As reported previously, insulin can suppress Cyp2e1 expression, whereas triiodothyronine (T₃) can upregulate Cyp2e1 expression [³⁰]. We first determined the blood glucose levels of the WT and Rdh13^−/− mice before and after CCl₄ treatment to exclude the possibility that altered insulin level affects Cyp2e1 expression. However, no significant difference was observed between the blood glucose levels of the WT and Rdh13^−/− mice (Fig. S4). Spot14 (thyroid hormone-inducible nuclear protein Spot14) is one of the T₃ downstream target genes [³¹]. Thus, we assessed the expression level of Spot14, indirectly representing the thyroxine metabolic status [³²]. We found that either Spot14 mRNA or protein levels in the livers of WT and Rdh13^−/− mice were induced after CCl₄ treatment. However, the induction of Spot14 in Rdh13^−/− mice was compromised at transcription (Fig. 5C) and protein (Fig. 5D) levels when compared with WT controls. Based on these data, the compromised response in Spot14 expression to CCl₄ may contribute to reduced Cyp2e1 expression levels in the absence of Rdh13.

Discussion

Rdh13 belongs to the SDR family and is localized in the mitochondria. As previously reported, Rdh13 is involved in vitamin A metabolism of humans and mice [¹,²]. Structurally, Rdh13 shows high-sequence similarity to Rdh11, Rdh12, and Rdh14, which are the members of the short-chain RDH family with dual-substrate specificity [⁶–⁸]. All the members of the RDH family are express in the mammalian retina and essential for the normal functioning of the retina [¹²,¹³]. In our previous study, by using Rdh13 knockout mouse model, we found that Rdh13 deficiency protects the retina from light damage [⁹]. Considering that Rdh13 is highly expressed in mouse liver, we explored the potential roles of Rdh13 in liver function. Unexpectedly, Rdh13 deficiency displayed no significant effect on the biochemical parameters of liver function and histological structure, suggesting a dispensable role of Rdh13 in the regulation of liver functions in mice. A compensative mechanism by other members of RDH family is triggered in the absence of Rdh13.

CCl₄-induced acute liver injury in mice opens a window to investigate the biological processes of liver injury and subsequent liver regeneration. Upon CCl₄ challenge, Rdh13^−/− mice were resistant to CCl₄ treatment, as suggested by its mildly elevated liver enzyme and less liver histological changes when compared with WT mice. The results of in situ TUNEL for apoptotic hepatocytes and immunostaining for caspase-3 expression also revealed that CCl₄-induced apoptosis cell is lesser in Rdh13^−/− mice than that in WT controls. In line with this data, the expression levels of antiapoptotic protein Bcl-2 and apoptotic protein Bax displayed downregulation and upregulation in the livers while lacking Rdh13 after CCl₄ treatment. We further evaluated the ability of hepatocyte compensatory proliferation following CCl₄-induced acute liver injury by Brdu incorporation. As expected, the percentage of Brdu-positive hepatocytes in Rdh13^−/− liver was lower than that in WT mice. Moreover, we showed that Rdh13^−/− mice displayed a weak response to Cyclin D1 expression, as well as ERK phosphorylation to CCl₄ treatment, implicating the involvement of Rdh13 in regulating ERK signaling.

Rdh13 may be involved in the metabolic conversion of CCl₄ by Cyp2e1 to CCl₃O and/or CCl₃OO radicals, which are toxic to mouse hepatocytes [³³]. This association may explain why Rdh13^−/− mice are resistant against CCl₄ toxicity. To this end, we investigated the mRNA and protein expression levels of Cyp2e1 and Spot14, indirectly reflecting the thyroxine metabolic status, because T₃ regulates Cyp2e1 expression in vivo [³⁴]. Our data indicated that Rdh13 deficiency reduced the expression levels of Cyp2e1 mRNA and protein in the liver tissues, especially at 48 h after CCl₄ treatment. Spot14 induction was also compromised in Rdh13^−/− livers to a greater degree than that in WT mice. Basing on these data, we concluded that Rdh13 participates in the regulation of thyroxine metabolism and contributes to the lowering of Cyp2e1 expression. These findings shed a light on the function of Rdh13 in liver disease in vivo and implicated Rdh13 as a potential new therapeutic target for the prevention and treatment of chemically induced liver injury.

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