Role of the Drug-Metabolizing Enzyme CYP during Mouse Liver Development

Abstract

The drug-metabolizing enzyme CYP is mainly involved in the metabolism of various substances in the liver, such as drugs, endogenous substances, and carcinogens. Recent reports have also revealed that CYP1B1 plays a major role in the developmental process. Because the level of CYP expression is markedly high in the liver, we hypothesize that CYP plays a role in the developmental process of the liver. To verify this hypothesis, we analyzed the expression patterns of various CYP molecular species and their functions during the differentiation of embryonic stem cells (ES cells) into hepatocytes and the developmental process in mice. The results demonstrated that CYP2R1 and CYP26A1 are expressed at an earlier stage of the differentiation of ES cells into hepatocytes than hepatoblast-specific markers. Additionally, during the development of the mouse liver, CYP2R1 and CYP26A1 were mostly up-regulated during the stage when hepatoblasts appeared. In addition, when CYP2R1 and CYP26A1 expressions were forced in ES cells and liver of adult mice, they differentiated into hepatoblast marker positive cells. These results suggest that CYP2R1 and CYP26A1 may play a major role in hepatoblast cell differentiation during the development of the liver.

The mature liver contains a variety of cell types, such as hepatocytes, sinusoidal endothelial cells, bile duct epithelial cells, stellate cells and Kupffer cells. The hepatocyte account for 80% of the volume of the liver and play a major role in liver functions, such as metabolism and excretion of drugs. Additionally, the fetal liver is known to act as a hematopoietic organ.

The expression of CYP is significantly up-regulated in the liver, and CYP converts fat-soluble drugs into a water-soluble state so that they can be easily excreted. In addition to drug metabolism, CYP is also known to be involved in the oxidative metabolism of endogenous substances, including steroids, bile acid, hormones, and eicosanoids. To date, 58 types of CYP molecular species have been identified in humans, and 108 types have been identified in mice.^1–3) As the CYP molecular species share high amino acid sequence homology in several substrate-recognition sites, in addition to the above-mentioned functions of CYP, it may also play a major role in development and differentiation in the body. Because CYP begins to be expressed close to the time of hematopoiesis initiation during the fetal stage and CYP requires heme for its structure, CYP is considered to be involved in the development of the liver during the fetal stage. Moreover, it has been reported that the metabolites of endogenous substances and the intermediate metabolites of chemical substances have an effect on the development of individuals and on homeostasis.^4–6) Therefore, CYP, which transiently appears during the process of development, is thought to possibly play an important role in the development of the liver.

It has been reported that the knockout of either the CYP26A1 gene in mice causes abnormal embryogeny resulting in embryonic lethal.⁷⁾ Mutation of the CYP1B1 gene is also known to cause primary congenital glaucoma in human beings.⁸⁾ The same trabecular defect in humans that causes primary congenital glaucoma was also observed in CYP1B1 and tyrosinase double-knockout mice.⁹⁾ These reports indicate that CYP not only works as a drug-metabolizing enzyme but also plays a major role in development. We focused our attention on the fact that the expression of CYP is much higher in the liver than other organs¹⁰⁾and hypothesized that CYP may play a role in hepatocyte differentiation and proliferation during liver development. In this study, we attempted to validate this hypothesis by using the differentiation induction system to differentiate embryonic stem (ES) cells into hepatocytes.¹¹⁾

ES cells are multipotent stem cells that can differentiate into various cell types through induction by humoral factors, such as cytokines.¹²⁾ ES cells also provide a useful means for analyzing the differentiation mechanism of different cell types by inducing ES cells to differentiate into target cell types.

MATERIALS AND METHODS

Animal Handling

Pregnancy ICR mice (E11, 14) were purchased from Japan SLC, Inc. (Tokyo Laboratory Animals Science Co., Ltd., Tokyo, Japan). The mice were kept at room temperature (r.t.) (24±1°C) and 55±5% humidity with 12 h of light (artificial illumination; 8:00–20:00). Food and water were available ad libitum. Each animal was used only once. The present study was conducted in accordance with the Guiding Principles for the Care and use of Laboratory Animals, as adopted by the Committee on Animal Research at Hoshi University.

Induction of Differentiation of ES Cells into Hepatocytes

Mouse ES cells (AES0125, Lot. 001) were purchased from the RIKEN BRC CELL BANK. Culture of the mouse ES cells was performed using a known methodology¹¹⁾ (Fig. 1). Frozen cells were thawed, dispersed at a density of 1×10⁶ cells/mL in culture medium, and seeded at 10 mL per 100 mm in a gelatin-coated dish.

Fig. 1. Schematic Diagram of the Protocol for Induction of Differentiation of ES Cells into Hepatocytes

The induction of the differentiation of ES cells into hepatocytes was performed using a known methodology. The ES cells were seeded at 2×10⁵ cells per 35 mm gelatin-coated dish or at 4×10⁴ cells per well on a Lab-Tek™ II Chamber Slide (Nunc, CA, U.S.A.) and were incubated at 37°C in 5% CO₂ for 72 h in stem medium (DS farmabiomedical, Osaka, Japan) (final conc. LIF: 1000 units/mL). The culture medium was then changed to stem medium containing LIF and 10⁻⁸ mol/L all-trans-retinoic acid (RA) (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the cells were incubated for 3 d.

After 3 d, the cells were cultured with LIF (−) culture medium for 5 d in the presence of fibroblast growth factor (FGF)-1, 100 ng/mL; FGF4, 20 ng/mL; hepatocyte growth factor (HGF), 50 ng/mL (Wako) at 10⁶ cells per 100 mm gelatin-coated dishes.

After 5 d, the cells were subcultured from the gelatin-coated dishes to collagen-coated dishes. At this point, the cells were washed gently three times with ES Cell Qualified Dulbecco’s phosphate buffered saline (D-PBS) and incubated with 1 mL of PBS for subculture at 37°C in 5% CO₂ for 2 min. The reaction was stopped by adding serum-free ES Cell Qualified Dulbecco’s modified Eagle’s medium (DMEM) (DS farmabiomedical), and the cells were collected and centrifuged at 100×g for 3 min. After removing the supernatant, the cells were resuspended in ES Cell Qualified DMEM and centrifuged at 100×g for 3 min, and the supernatant was removed. The cells were then resuspended in stem medium, seeded at 2×10⁵ cells per 35 mm collagen-coated dish and incubated in stem medium containing oncostatin M (OsM) (Wako) (10 ng/mL) for 2 d. After two days, the culture medium was changed from stem medium to hepatocyte medium(William’s E medium (Wako) containing insulin (Wako) 5 µg/mL, transferrin (Wako) 5 µg/mL, Bovine Serum Albumin (Wako) 0.5 mg/mL, ascorbic acid (Wako) 2 µmol/L, hydrocortisone-21-hemisuccinate (Wako) 10⁻² M) and the cells were incubated for 8 d.

Extraction of Total RNA

Total RNA was extracted from the cells during the induction of the differentiation of ES cells into hepatocytes using TRI reagent (Sigma-Aldrich, St. Louis, MO, U.S.A.).

cDNA was synthesized from 1 µg purified total RNA using a High Capacity cDNA synthesis Kit (Applied Biosystems, Foster City, CA, U.S.A.). The concentration of the total RNA (µg/mL) was calculated, and the purity of the total RNA was evaluated by measuring its absorbance at 260 and 280 nm.

For each sample, 2.0 µL of 10×RT buffer, 0.8 µL of 25×deoxynucleotide triphosphate (dNTP) Mix, 2.0 µL of 10×RT Random Primer, 1.0 µL MultiScribe™ Reverse Transcriptase, 1.0 µL ribonuclease (RNase) Inhibitor, and 3.2 µL ultrapure water, which were all included in the High Capacity cDNA synthesis Kit, were mixed gently on ice to prepare the 2×Reverse Transcription (RT) Master Mix.

RT-PCR

The following reagents were added to each well of the PCR 8 Strip Tube: 0.1 µL TaKaRa Ex Taq (TaKaRa-Bio, Shiga, Japan), 2.5 µL of 10×Ex Taq buffer, 2.0 µL dNTP mixture, 1.25 µL dimethyl sulfoxide (DMSO), 1 µL cDNA solution, 2.5 µL forward primer (20 pmol/µL), 2.5 µL reverse primer (20 pmol/µL) and 13.15 µL ultrapure water. Using an iQ™ Thermal Cycler (Bio-Rad, Hercules, CA, U.S.A.), samples were first denatured at 94°C for 2 min and then at 98°C for 10 s; then primer annealing was performed at 55 to 58°C for 30 s, followed by elongation at 72°C for 30 s. These steps constituted one cycle. After 30 to 35 cycles, an extension step was performed at 72°C for 1 min and 30 s to amplify the cDNA.

The forward and reverse primers for the hepatocellular differentiation markers, hepatocyte nuclear factor 3-beta (HNF-3β), α-fetoprotein (AFP), delta-like 1 (DLK1), albumin (ALB), and tryptophan dioxygenase (TDO), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene are listed in Table 1. After the PCR was completed, 2.5 µL of 10× loading buffer was added to the 25 µL PCR products, and the solution was mixed well. Agarose gel electrophoresis was performed with a 1.5% agarose gel and Tris acetate EDTA (TAE) buffer with 15 µL of PCR product/lane at r.t. for 30 min (Mupid-2 plus, TaKaRa-Bio). After electrophoresis, the agarose gel was soaked in EtBr solution (Nippon Gene, Tokyo, Japan), a nucleic acid stain solution, in the dark at r.t. for 15 min. The agarose gel was photographed using a cooled CCD camera (LAS-3000mini, FUJIFILM Corporation, Tokyo, Japan).

Table 1. Primers and Conditions for RT-PCR

Target gene	Accession No.	Primers	Forward:	Product size (bp)	Annealing temperature (°C)	Cycles
			Reverse:
HNF-3ß	NM_010446	For: 5′-ACTGGAGCAGCTACTACG-3′		169	58	30
		Rev: 5′-CCCACATAGGATGACATG-3′
AFP	NM_007423	For: 5′-CACTGCTGCAACTCTTCGTA-3′		300	58	30
		Rev: 5′-CTTTGGACCCTCTTCTGTGA-3′
DLK1	NM_007865	For: 5′-ATGCTTCCTGCCTGTGC-3′		245	55	35
		Rev: 5′-GCACGGGCCACTGGC-3′
ALB	NM_009654	For: 5′-TGAACTGGCTGACTGCTGTG-3′		718	58	32
		Rev: 5′-CATCCTTGGCCTCAGCATAG-3′
TDO	NM_019911	For: 5′-AGAGCCAGCAAAGGAGGAC-3′		500	55	35
		Rev: 5′-CTGTCTGCTCCTGCTCTGAT-3′
CYP3A11	NM_ 007818	For: 5′-CGCCTCTCCTTGCTGTCACA-3′		260	55	32
		Rev: 5′-CTTTGCCTTCTGCCTCAAGT-3′
CYP26A1	NM_007811	For: 5′-ACATTGCAGATGGTGCTTCA-3′		376	58	32
		Rev: 5′-TCACCTCGGGGTAGACCA-3′
CYP2 R I	NM_ 177382	For: 5′-CCATGGATTGGCATCTTACC-3′		361	58	30
		Rev: 5′-CCCAAGAAGGTCTCCTGTTG-3′
GAPDH	NM_002046	For: 5′-ATGGGGAAGGTGAAGGTCG-3′		177	58	30
		Rev: 5′-GGGGTCATIGATGGCAACAATA-3′

Immunocytochemistry

After washing with PBS(−), ES cells were fixed with 4% paraformaldehyde (PFA)/PBS at r.t. for 10 min. After washing gently with PBS(−) once, the cells were incubated with 0.05% TritonX-100/5% FCS /PBS at r.t. for 1 h and then with the primary antibody (Ab) solution at 4°C overnight. After washing gently with PBS(−) three times, the cells were reacted with the secondary Ab solution in the dark at r.t. for 1 h. After washing gently with PBS(−) three times, the cells were enclosed with VECTASHIELD (Vector Laboratories, Bulingama, CA, U.S.A.) using MICRO COVER GLASS. The immunostained sections were detected using FV-1200 (Olympus). Anti-mouse CYP2R1 (AP7894c) was purchased from ABGENT, Inc. (San Diego, CA, U.S.A.). Anti-mouse CYP26A1 (ab64888) was purchased from Abcam plc. Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG) (A21206) was purchased from Invitrogen.

Immunohistochemistry

ICR pregnant mice at 9 and 11 d of gestation were anesthetized with pentobarbital via intraperitoneal injection, and fetuses were removed by cesarean section. The fetuses were fixed in 4% PFA at 4°C for 20 min. After washing with PBS(−), the samples were soaked in 10, 20, and 30% sucrose solutions at 4°C in sequence until the tissues sank in each solution. The samples were embedded in O.C.T compound and stored at −80°C. Then, they were sectioned into 12-µm slices using LEICA CM1850 and placed on micro slide glass. Once the sections were sufficiently dried, they were stored at −80°C as frozen sections until use. Immunostaining was then performed in the same manner as immunocytochemistry.

Plasmid DNA

pEF-BOS were by inserting the EcoRI/SalI mouse CYP2R1 and CYP26A1 cDNA fragment amplified by PCR into the EcoRI/SalI site of pEF-BOS. pEF-BOS-IRES-GFP was constructed by inserting the green fluorescent protein (GFP). To estimate the transfected cells, we constructed pEF-BOS-IRES-GFP containing a GFP driven by an internal ribosomal entry site (IRES). CYP2R1 Forward Primer: 5′-GAA TTC TAC CAT GTT GGA GCT ACC GGG AGC CCG-3′, Reverse Primer: 5′-GTC GAC TCA GCG TCT TTC TGC ACA GAT GA-3′, CYP26A1 Forward Primer: 5′-GAA TTC TAC CAT GGG GCT CCC GGC GCT GCT GG-3′, Reverse Primer: 5′-GTC GAC TCA GAT ATC TCC CTG GAA GT-3′

In Vitro Transfection

On the day after seeding at 1×10⁴ cells per well on 8-well chamber slides (Nunc), 0.5 µg of pEF-BOS-IRES-enhanced green fluorescent protein (EGFP) (vehicle), pEF-BOS-CYP2R1-IRES-EGFP or pEF-BOS-CYP26A1-IRES-EGFP, 50 µL of Opti-MEM solution, and 1 µL of TranIT-LT1 reagent were mixed and incubated at r.t. for 15 min; then, the mixture was added to the cells.

In Vivo Transfection

For in vivo transfection, 2.1 mL TransIT-EE Hydrodynamic Delivery Solution (Mirus Bio) and 40 µg/100 µL each of pEF-BOS-IRES-EGFP (vehicle), pEF-BOS-CYP2R1-IRES-EGFP, and pEF-BOS-CYP26A1-IRES-EGFP were mixed, and the entire solution (2.1 mL) was administered to 4-week-old mice by rapid tail vein injection. Three days after the mouse was transfected with the expression vector, its abdomen was opened under pentobarbital anesthesia. After the heart was washed with PBS to remove blood and perfusion fixation was performed with 4% PFA, the liver of the mouse was removed.

RESULTS

Expression Patterns of CYPs mRNA during the Induction of the Differentiation of ES Cells into Hepatocytes

We analyzed the mRNA expression patterns of CYP molecular species that appeared during the induction of the differentiation of ES cells into hepatocytes (Fig. 2). The results showed that hepatocyte nuclear factor 3-beta (HNF-3β), which indicates differentiation of ES cells into endodermal cells, was first expressed on day 3 of differentiation induction, and its expression was highest on day 7. For α-fetoprotein (AFP) and delta-like-1 (DLK1), which are hepatoblast markers, expression was first observed on day 7 of differentiation induction. Albumin (ALB), which is expressed at the late stage of hepatocyte differentiation, was first expressed on day 11 of differentiation induction. The expression of glucose-6-phosphate (G6Pase), a marker of adult hepatocytes, was first observed on day 7 of differentiation induction, while that of Trp dioxygenase (TDO) was only observed on day 18 (Fig. 2). On day 18, the cells were also positive for periodic acid-Schiff (PAS) staining, which is an indicator of the ability of cells to store glycogen (data not shown).

Fig. 2. Patterns of mRNA Expression of CYP Molecular Species during the Induction of Differentiation of ES Cells into Hepatocytes

ES cells were differentiated into hepatocytes by induction, and cells were collected on Days 0 (undifferentiated ES), 3, 7, 11 and 18 of differentiation induction. After reverse transcription, the mRNA expression levels of drug-metabolizing enzymes and markers of hepatocellular differentiation were analyzed by RT-PCR.

The most important role of CYP3A11 is its drug-metabolizing enzyme function, and CYP3A11 was first expressed on day 11 of differentiation induction. Thereafter, the expression level of CYP3A11 increased with the maturation of the hepatocytes. CYP2R1 was first expressed on day 3 of differentiation induction, and its expression was highest on day 7, after which it decreased with further differentiation induction. In addition, high expression of CYP26A1 was observed on day 3 of hepatocyte differentiation induction; its expression was decreased on day 7 before being highly expressed again on day 18 (Fig. 2). Intriguingly, CYP2R1 and CYP26A1 were both induced prior to AFP and DLK1, which are makers of hepatoblasts.

Expression Patterns of CYP2R1 and CYP26A1 during the Induction of the Differentiation of ES Cells into Hepatocytes

mRNA expression of CYP2R1 and CYP26A1 was observed prior to the differentiation of ES cells into hepatoblasts. Therefore, to evaluate whether both CYPs were also expressed at the protein level at that time, immunostaining was performed using anti-CYP2R1 Ab or anti-CYP26A1 Ab and an Ab for delta-like 1 (DLK-1), a marker of hepatoblasts. As a result, on day 3 of differentiation induction, when ES cells had differentiated into endoderm, although CYP2R1 and CYP26A1 were already expressed, the expression of DLK1, a marker of hepatoblasts, was not observed (Fig. 3). On day 7, many cells expressing CYP2R1 or CYP26A1 were positive for DLK1 (Fig. 3).

Fig. 3. Patterns of Expression of CYP26A1 or CYP2R1 during the Induction of the Differentiation of ES Cells into Hepatoblasts

Cells on Days 3 and 7 of differentiation induction of ES cells into hepatocytes were fixed, and the expression patterns of CYP2R1 or CYP26A1 were analyzed by immunostaining.

Based on these results, it is possible that CYP2R1 and CYP26A1 are expressed prior to hepatoblast markers during hepatocyte differentiation and that they play a particular roles in the process of differentiation of ES cells into hepatoblasts.

Expression Pattern of CYP2R1 and CYP26A1 in the Fetal Liver

During the development of the mouse liver, the development of the hepatic primordium of fetal mice at E8 to E13 days of gestation was considered to correspond to the differentiation of endoderm to hepatoblasts. Therefore, we conducted immunohistochemical staining of the livers of fetal mice at E9 and E11 days of gestation to analyze whether CYP2R1 and CYP26A1 were expressed at the stage of hepatoblast appearance.

The results revealed that CYP2R1 and CYP26A1 were expressed in the hepatic primordium of fetal mice at E9 days of gestation and that some of the CYP2R1-positive cells and CYP26A1-positive cells were also positive for DLK1 (Fig. 4). Furthermore, many cells in the liver of fetal mice at E11 days of gestation were positive for both CYP2R1 and CYP26A1, and a significant number of them also expressed DLK1 (Fig. 4).

Based on these results, the probable involvement of CYP2R1 and CYP26A1 in the differentiation of hepatoblasts was also confirmed in the process of mouse liver development.

Fig. 4. Patterns of Expression of CYP26A1 and CYP2R1 in the Fetal Liver

Frozen sections of the liver of fetal mice at days 9 and 11 of gestation were made, and the expression patterns of CYP2R1 and CYP26A1 in the hepatic primordium were analyzed by immunohistochemical staining.

Induction of Delta-Like 1-Positive Cells by Forced Expression of CYP2R1 and CYP26A1 in ES Cells

Because the involvement of CYP2R1 and CYP26A1 in the differentiation of hepatoblasts was indicated by the results of our above experiments, to further verify this finding, we investigated whether forced expression of CYP2R1 or CYP26A1 in ES cells would produce DLK1-positive cells. Expression plasmids encoding CYP2R1 or CYP26A1 were transfected into ES cells, and differentiated state of the cells were examined at 72 h after transfection. Our results showed that GFP expressing cells (expressing CYP2R1 or CYP26A1) differentiated into hepatoblasts that were positive for DLK1 (Fig. 5).

Fig. 5. Forced Expression of CYP26A1 and CYP2R1 in ES Cells

Vehicle, CYP2R1 and CYP26A1 were expressed in ES cells. The cells were incubated for 72 h and immunostained with DLK1 Ab and anti-GFP Ab.

Forced Expression of CYP2R1 and CYP26A1 in Mouse Liver Induces DLK1-Positive Cells

In the in vitro experiments, it was revealed that forced expression of CYP2R1 or CYP26A1 caused differentiation of ES cells into hepatoblasts. Thus, in this study, we investigated whether forced expression of CYP2R1 and CYP26A1 would differentiate into hepatoblasts in mouse liver. For gene transfer to the liver, mice received a tail vein injection of plasmid DNA (Vehicle, CYP2R1, CYP26A1) dissolved in a hydrodynamic solution. The results showed that GFP expressing cells differentiated into DLK1-positive cells (Fig. 6).

Fig. 6. Forced Expression of CYP2R1 and CYP26A1 in Mouse Liver

Vehicle, CYP2R1 and CYP26A1 expression vectors were administered by tail vein injection, and 72 h later, liver tissue sections were obtained and immunostained with anti-EGFP and anti-DLK1 antibodies.

DISCUSSION

To efficiently induce differentiation of ES cells into hepatocytes, it is essential to cause differentiation of ES cells into endodermal cells and then further differentiation and maturation specifically into hepatocytes. It has been reported that when ES cells are incubated with retinoic acid (RA) in the presence of leukemia inhibitory factor (LIF), most cells differentiate into endodermal cells.¹³⁾ The factors that were added during the process of differentiation induction from endodermal cells to hepatocytes are the factors secreted during the development of hepatoblast tissues. The budding of hepatoblast tissue is initiated by stimulation by fibroblast growth factor (FGF) secreted by the adjacent cardiac mesoderm and bone morphogenic protein (BMP) secreted by the transverse septum.¹⁴⁾ Thereafter, maturation of hepatocytes is promoted by adding hepatocyte growth factor (HGF) and oncostatin M (OsM), which are involved in the maturation of fetal hepatocytes. ES cells were reported to be efficiently differentiated into hepatocytes by the addition of a combination of FGF-1, FGF-4, and HGF, which are molecules that are increased during hepatic damage, to the culture medium.¹¹⁾

This study used the differentiation induction system to differentiate ES cells into hepatocytes to examine CYP molecular species that contribute to the proliferation and differentiation of the fetal liver. In the process of differentiation induction from ES cells into hepatocytes, the CYP molecular species that showed interesting expression patterns were CYP2R1 and CYP26A1. CYP2R1 and CYP26A1 are involved in the metabolic pathways of vitamin A and vitamin D, respectively.

CYP26A1 contributes to the synthesis and metabolism of retinoic acid. β-Carotene and retinyl ether are converted to retinol (alcohol) and subsequently oxidized to retinal (aldehyde) and then retinoic acid, which has physiological action after it undergoes further oxidization.¹⁵⁾ It is known that the metabolism of retinoic acid involves CYP26 family members in the embryonic stage and CYP3A7 in the fetal stage to regulate the concentration of retinoic acid. Retinoic acid contributes to development and differentiation by binding to nuclear receptors, such as retinoic acid receptor (RAR) and retinoid X receptor (RXR).¹⁶⁾ Vitamin A is the collective name for retinal, retinol and retinoic acid and is associated with the differentiation, proliferation, morphogenesis, and apoptosis of cells. Because an excessive intake of vitamin A also causes teratogenicity, regulation of the concentration of retinoic acid is highly important for the living body. In CYP26A1 knockout mice, which show embryonic lethal or death shortly after birth, morphological defects are particularly observed in the tailbud and hindbrain, likely because CYP26A1 expression is localized there.⁷⁾ Thus, in CYP26A1 knockout mice, the concentration of retinoic acid is elevated in those areas, resulting in the development of hypoplasia in the lower part of the body, which generates a mermaid-like morphology. The results of this study showed that CYP26A1 was first expressed on day 3 of differentiation induction. Because retinoic acid had been added to the culture medium until day 3, it was suggested that the expression of CYP26A1 was possibly induced to metabolize that retinoic acid to control its concentration.

The expression of both CYP2R1 and CYP26A1 was observed in the period of day 3 and 7 in this culture system, which is the period of differentiation from an endoderm to a hepatoblast. This day 3 and 7 period of ES cells is considered to be equal to Embryonic day of 8 and 13. At the onset of liver development at approximately mouse embryonic day (E9). Therefore, we could not isolate liver alone in this embryonic stage (E8) and analyzed the hepatic primordium (also called liver bud) in E9.

CYP26A1 was also shown to be expressed in the hepatic primordium of fetal mice at E9 to E11 days of gestation (Fig. 4). In recent years, the ability of primary hepatocytes from mice at E14.5 d of gestation to store glycogen has been reported to be increased by the addition to culture medium of all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9CRA), ligands of RAR and RXR, respectively.¹⁷⁾ Based on these findings, CYP26A1 expression in hepatoblasts was suggested to be involved in the differentiation of endoderm cells into hepatoblasts by binding to nuclear receptors, such as RAR and RXR, resulting in the regulation of the concentrations of ATRA and 9CRA in the living body.

CYP2R1 is expressed in the liver and testes of mice and catalyzes 25-hydroxylation of vitamin D3.¹⁰⁾ To date, at least 6 enzymes, including CYP2C11, CYP2D25, CYP2J3, CYP2R1, CYP3A4, and CYP27A1, have been reported to be responsible for 25-hydroxylation, and CYP2R1, which has the common substrates vitamin D2 and D3, is universally found in a variety of species.¹⁰⁾ The amino acid sequence of CYP2R1 is conserved in various species, and mice and humans share 89% homology in the CYP2R1 sequence. Active vitamin D receptor (VDR) contributes to the regulation of the expression of E-cadherin and plays an important role in intercellular communication through E-cadherin.¹⁸⁾ Beta-catenin binds to the intracellular domain of E-cadherin and is known to be a regulator of gene transcription. Beta-catenin has been reported to be transiently expressed during liver development.¹⁹⁾ Additionally, knockout of the β-catenin gene in mouse hepatoblasts has been shown to decrease cellular proliferative potency, resulting in embryonic lethal.²⁰⁾ Based on these reports, the transient expression of CYP2R1 may contribute to the proliferation of hepatoblasts via VDR. In this study, a pattern of expression of CYP2R1 was observed: its expression increased from days 3 to 7 of differentiation induction and was reduced thereafter. Therefore, it was suggested that CYP2R1 may play an important role in the early process of differentiation of ES cells into hepatocytes (hepatoblasts).

Transfection assay for in ES cells using an expression vector of CYP2R1 or CYP26A1 resulted in DLK1-positive cell induction (Fig. 5); however, it remains unknown whether CYP2R1 and CYP26A1 regulate the differentiation of ES cells into hepatocytes by metabolizing vitamin D and/or vitamin A in the culture medium or endogenous vitamin D and/or vitamin A. Furthermore, in the in vivo forced expression experiments of CYP2R1 and CYP26A1, it was unclear which cell types were transfected (Fig. 6). Therefore, it is unknown whether undifferentiated hepatic stem cells or adult hepatocytes differentiated into hepatoblasts. To clarify the mechanisms of the development and regeneration of the liver, detailed analyses of the functions of CYP2R1 and CYP26A1 are necessary.

In this study, in both the induction of differentiation of ES cells into hepatocytes experiments and the fetal mice experiments, the expressions of CYP2R1 and CYP26A1 were induced prior to the expression of DLK1, a hepatoblast marker. These results suggest the involvement of these CYPs in hepatoblast differentiation through the metabolism of endogenous substances, such as vitamins D and A.

Uncovering the mechanism of CYP2R1 and CYP26A1 expression regulation will likely help clarify the mechanism of early liver development and/or liver regeneration.

Acknowledgments

The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan-Supported Program for the Strategic Research Foundation at Private Universities, 2014–2018, S1411019. We thank Ms. Haruka Kato, Ms. Misa Iizuka, Ms. Konomi Oba, Ms. Mami Nakai, Ms. Saori Tomita, Ms. Tomoka Yasukawa, for their technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

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