Vol. 39, No. 12 Biol. Pharm. Bull. 39, 2015–2021 (2016) 2015 Regular Article

Role of the Drug-Metabolizing CYP during Mouse Liver Development Wataru Ochiai,# Akiyo Hirose,# Taisuke Kawamura, Kyoko Komachi, Yuka Yamamoto, Satoshi Kitaoka, Jo Hatogai, Yoshiki Kusunoki, Risako Kon, Nobutomo Ikarashi, and Kiyoshi Sugiyama* Department of Clinical Pharmacokinetics, School of Pharmacy and Pharmaceutical Sciences, Hoshi University; Tokyo 142–8501, Japan. Received June 15, 2016; accepted August 23, 2016

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 hypoth- esis, 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. Key words embryonic stem cell; liver; CYP; hepatoblast

The mature liver contains a variety of cell types, such as ing in embryonic lethal.7) Mutation of the CYP1B1 is hepatocytes, sinusoidal endothelial cells, bile duct epithelial also known to cause primary congenital glaucoma in human cells, stellate cells and Kupffer cells. The hepatocyte account beings.8) The same trabecular defect in humans that causes for 80% of the volume of the liver and play a major role in primary congenital glaucoma was also observed in CYP1B1 liver functions, such as metabolism and excretion of drugs. and tyrosinase double-knockout mice.9) These reports indicate Additionally, the fetal liver is known to act as a hematopoietic that CYP not only works as a drug-metabolizing enzyme but organ. also plays a major role in development. We focused our atten- The expression of CYP is significantly up-regulated in the tion on the fact that the expression of CYP is much higher in liver, and CYP converts fat-soluble drugs into a water-soluble the liver than other organs10)and hypothesized that CYP may state so that they can be easily excreted. In addition to drug play a role in hepatocyte differentiation and proliferation dur- metabolism, CYP is also known to be involved in the oxida- ing liver development. In this study, we attempted to validate tive metabolism of endogenous substances, including , this hypothesis by using the differentiation induction system bile acid, hormones, and eicosanoids. To date, 58 types of to differentiate embryonic stem (ES) cells into hepatocytes.11) CYP molecular species have been identified in humans, and ES cells are multipotent stem cells that can differentiate 108 types have been identified in mice.1–3) As the CYP mo- into various cell types through induction by humoral factors, lecular species share high amino acid in such as cytokines.12) ES cells also provide a useful means for several substrate-recognition sites, in addition to the above- analyzing the differentiation mechanism of different cell types mentioned functions of CYP, it may also play a major role in by inducing ES cells to differentiate into target cell types. development and differentiation in the body. Because CYP begins to be expressed close to the time of hematopoiesis MATERIALS AND METHODS initiation during the fetal stage and CYP requires heme for its structure, CYP is considered to be involved in the develop- Animal Handling Pregnancy ICR mice (E11, 14) were ment of the liver during the fetal stage. Moreover, it has been purchased from Japan SLC, Inc. (Tokyo Laboratory Animals reported that the metabolites of endogenous substances and Science Co., Ltd., Tokyo, Japan). The mice were kept at room the intermediate metabolites of chemical substances have an temperature (r.t.) (24±1°C) and 55±5% humidity with 12 h of effect on the development of individuals and on homeosta- light (artificial illumination; 8:00–20:00). Food and water were sis.4–6) Therefore, CYP, which transiently appears during the available ad libitum. Each animal was used only once. The process of development, is thought to possibly play an impor- present study was conducted in accordance with the Guid- tant role in the development of the liver. ing Principles for the Care and use of Laboratory Animals, It has been reported that the knockout of either the as adopted by the Committee on Animal Research at Hoshi CYP26A1 gene in mice causes abnormal embryogeny result- University. Induction of Differentiation of ES Cells into Hepatocytes # These authors contributed equally to this work. Mouse ES cells (AES0125, Lot. 001) were purchased from the

* To whom correspondence should be addressed. e-mail: [email protected] © 2016 The Pharmaceutical Society of Japan 2016 Biol. Pharm. Bull. Vol. 39, No. 12 (2016)

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

RIKEN BRC CELL BANK. Culture of the mouse ES cells MO, U.S.A.). was performed using a known methodology11) (Fig. 1). Frozen cDNA was synthesized from 1 µg purified total RNA using cells were thawed, dispersed at a density of 1×106 cells/mL in a High Capacity cDNA synthesis Kit (Applied Biosystems, culture medium, and seeded at 10 mL per 100 mm in a gelatin- Foster City, CA, U.S.A.). The concentration of the total RNA coated dish. (µg/mL) was calculated, and the purity of the total RNA was The induction of the differentiation of ES cells into hepa- evaluated by measuring its absorbance at 260 and 280 nm. tocytes was performed using a known methodology. The ES For each sample, 2.0 µL of 10×RT buffer, 0.8 µL of 25×de- cells were seeded at 2×105 cells per 35 mm gelatin-coated dish oxynucleotide triphosphate (dNTP) Mix, 2.0 µL of 10×RT or at 4×104 cells per well on a Lab-Tek™ II Chamber Slide Random Primer, 1.0 µL MultiScribe™ Reverse Transcriptase,

(Nunc, CA, U.S.A.) and were incubated at 37°C in 5% CO2 1.0 µL ribonuclease (RNase) Inhibitor, and 3.2 µL ultrapure for 72 h in stem medium (DS farmabiomedical, Osaka, Japan) water, which were all included in the High Capacity cDNA (final conc. LIF: 1000 units/mL). The culture medium was synthesis Kit, were mixed gently on ice to prepare the 2×Re- then changed to stem medium containing LIF and 10−8 mol/L verse Transcription (RT) Master Mix. all-trans-retinoic acid (RA) (Wako Pure Chemical Industries, RT-PCR The following reagents were added to each well Ltd., Osaka, Japan), and the cells were incubated for 3 d. of the PCR 8 Strip Tube: 0.1 µL TaKaRa Ex Taq (TaKaRa-Bio, After 3 d, the cells were cultured with LIF (−) culture Shiga, Japan), 2.5 µL of 10×Ex Taq buffer, 2.0 µL dNTP mix- medium for 5 d in the presence of fibroblast growth factor ture, 1.25 µL dimethyl sulfoxide (DMSO), 1 µL cDNA solu- (FGF)-1, 100 ng/mL; FGF4, 20 ng/mL; hepatocyte growth fac- tion, 2.5 µL forward primer (20 pmol/µL), 2.5 µL reverse prim- tor (HGF), 50 ng/mL (Wako) at 106 cells per 100 mm gelatin- er (20 pmol/µL) and 13.15 µL ultrapure water. Using an iQ™ coated dishes. Thermal Cycler (Bio-Rad, Hercules, CA, U.S.A.), samples After 5 d, the cells were subcultured from the gelatin-coated were first denatured at 94°C for 2 min and then at 98°C for dishes to collagen-coated dishes. At this point, the cells were 10 s; then primer annealing was performed at 55 to 58°C for washed gently three times with ES Cell Qualified Dulbecco’s 30 s, followed by elongation at 72°C for 30 s. These steps con- phosphate buffered saline (D-PBS) and incubated with 1 mL stituted one cycle. After 30 to 35 cycles, an extension step was of PBS for subculture at 37°C in 5% CO2 for 2 min. The re- performed at 72°C for 1 min and 30 s to amplify the cDNA. action was stopped by adding serum-free ES Cell Qualified The forward and reverse primers for the hepatocellular Dulbecco’s modified Eagle’s medium (DMEM) (DS farma- differentiation markers, hepatocyte nuclear factor 3-beta biomedical), and the cells were collected and centrifuged at (HNF-3β), α-fetoprotein (AFP), delta-like 1 (DLK1), albumin 100×g for 3 min. After removing the supernatant, the cells (ALB), and tryptophan dioxygenase (TDO), and glyceralde- were resuspended in ES Cell Qualified DMEM and centri- hyde-3-phosphate dehydrogenase (GAPDH) as the housekeep- fuged at 100×g for 3 min, and the supernatant was removed. ing gene are listed in Table 1. After the PCR was completed, The cells were then resuspended in stem medium, seeded at 2.5 µL of 10× loading buffer was added to the 25 µL PCR 2×105 cells per 35 mm collagen-coated dish and incubated in products, and the solution was mixed well. Agarose gel elec- stem medium containing oncostatin M (OsM) (Wako) (10 ng/ trophoresis was performed with a 1.5% agarose gel and Tris mL) for 2 d. After two days, the culture medium was changed acetate EDTA (TAE) buffer with 15 µL of PCR product/lane from stem medium to hepatocyte medium(William’s E me- at r.t. for 30 min (Mupid-2 plus, TaKaRa-Bio). After electro- dium (Wako) containing insulin (Wako) 5 µg/mL, transferrin phoresis, the agarose gel was soaked in EtBr solution (Nippon (Wako) 5 µg/mL, Bovine Serum Albumin (Wako) 0.5 mg/mL, Gene, Tokyo, Japan), a nucleic acid stain solution, in the dark ascorbic acid (Wako) 2 µmol/L, hydrocortisone-21-hemisucci- at r.t. for 15 min. The agarose gel was photographed using a −2 nate (Wako) 10 M) and the cells were incubated for 8 d. cooled CCD camera (LAS-3000mini, FUJIFILM Corporation, Extraction of Total RNA Total RNA was extracted from Tokyo, Japan). the cells during the induction of the differentiation of ES cells Immunocytochemistry After washing with PBS(−), ES into hepatocytes using TRI reagent (Sigma-Aldrich, St. Louis, cells were fixed with 4% paraformaldehyde (PFA)/PBS at r.t. Vol. 39, No. 12 (2016) Biol. Pharm. Bull. 2017

Table 1. Primers and Conditions for RT-PCR

Forward: Target gene Accession No. Primers 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′ for 10 min. After washing gently with PBS(−) once, the cells GAC TCA GAT ATC TCC CTG GAA GT-3′ were incubated with 0.05% TritonX-100/5% FCS /PBS at r.t. In Vitro Transfection On the day after seeding at for 1 h and then with the primary antibody (Ab) solution at 1×104 cells per well on 8-well chamber slides (Nunc), 0.5 µg 4°C overnight. After washing gently with PBS(−) three times, of pEF-BOS-IRES-enhanced green fluorescent the cells were reacted with the secondary Ab solution in the (EGFP) (vehicle), pEF-BOS-CYP2R1-IRES-EGFP or pEF- dark at r.t. for 1 h. After washing gently with PBS(−) three BOS-CYP26A1-IRES-EGFP, 50 µL of Opti-MEM solution, times, the cells were enclosed with VECTASHIELD (Vector and 1 µL of TranIT-LT1 reagent were mixed and incubated at Laboratories, Bulingama, CA, U.S.A.) using MICRO COVER r.t. for 15 min; then, the mixture was added to the cells. GLASS. The immunostained sections were detected using In Vivo Transfection F o r in vivo transfection, 2.1 mL FV-1200 (Olympus). Anti-mouse CYP2R1 (AP7894c) was TransIT-EE Hydrodynamic Delivery Solution (Mirus Bio) purchased from ABGENT, Inc. (San Diego, CA, U.S.A.). Anti- and 40 µg/100 µL each of pEF-BOS-IRES-EGFP (vehicle), mouse CYP26A1 (ab64888) was purchased from Abcam plc. pEF-BOS-CYP2R1-IRES-EGFP, and pEF-BOS-CYP26A1- Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (IgG) IRES-EGFP were mixed, and the entire solution (2.1 mL) was (A21206) was purchased from Invitrogen. administered to 4-week-old mice by rapid tail vein injection. Immunohistochemistry ICR pregnant mice at 9 and 11 d Three days after the mouse was transfected with the expres- of gestation were anesthetized with pentobarbital via intra- sion vector, its abdomen was opened under pentobarbital anes- peritoneal injection, and fetuses were removed by cesarean thesia. After the heart was washed with PBS to remove blood section. The fetuses were fixed in 4% PFA at 4°C for 20 min. and perfusion fixation was performed with 4% PFA, the liver After washing with PBS(−), the samples were soaked in 10, of the mouse was removed. 20, and 30% sucrose solutions at 4°C in sequence until the tissues sank in each solution. The samples were embedded RESULTS in O.C.T compound and stored at −80°C. Then, they were sectioned into 12-µm slices using LEICA CM1850 and placed Expression Patterns of CYPs mRNA during the Induc- on micro slide glass. Once the sections were sufficiently dried, tion of the Differentiation of ES Cells into Hepatocytes they were stored at −80°C as frozen sections until use. Im- We analyzed the mRNA expression patterns of CYP mo- munostaining was then performed in the same manner as im- lecular species that appeared during the induction of the dif- munocytochemistry. ferentiation of ES cells into hepatocytes (Fig. 2). The results Plasmid DNA pEF-BOS were by inserting the EcoRI/SalI showed that hepatocyte nuclear factor 3-beta (HNF-3β), which mouse CYP2R1 and CYP26A1 cDNA fragment amplified by indicates differentiation of ES cells into endodermal cells, was PCR into the EcoRI/SalI site of pEF-BOS. pEF-BOS-IRES- first expressed on day 3 of differentiation induction, and its GFP was constructed by inserting the green fluorescent protein expression was highest on day 7. For α-fetoprotein (AFP) and (GFP). To estimate the transfected cells, we constructed pEF- delta-like-1 (DLK1), which are hepatoblast markers, expres- BOS-IRES-GFP containing a GFP driven by an internal ribo- sion was first observed on day 7 of differentiation induction. somal entry site (IRES). CYP2R1 Forward Primer: 5′-GAA Albumin (ALB), which is expressed at the late stage of he- TTC TAC CAT GTT GGA GCT ACC GGG AGC CCG-3′, patocyte differentiation, was first expressed on day 11 of dif- Reverse Primer: 5′-GTC GAC TCA GCG TCT TTC TGC ACA ferentiation induction. The expression of glucose-6-phosphate GAT GA-3′, CYP26A1 Forward Primer: 5′-GAA TTC TAC CAT (G6Pase), a marker of adult hepatocytes, was first observed GGG GCT CCC GGC GCT GCT GG-3′, Reverse Primer: 5′-GTC on day 7 of differentiation induction, while that of Trp dioxy- 2018 Biol. Pharm. Bull. Vol. 39, No. 12 (2016)

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 and markers of hepatocellular differentiation were analyzed by RT-PCR. genase (TDO) was only observed on day 18 (Fig. 2). On day Expression Pattern of CYP2R1 and CYP26A1 in the 18, the cells were also positive for periodic acid-Schiff (PAS) Fetal Liver During the development of the mouse liver, staining, which is an indicator of the ability of cells to store the development of the hepatic primordium of fetal mice at glycogen (data not shown). E8 to E13 days of gestation was considered to correspond to The most important role of CYP3A11 is its drug-metabo- the differentiation of endoderm to hepatoblasts. Therefore, lizing enzyme function, and CYP3A11 was first expressed on we conducted immunohistochemical staining of the livers of day 11 of differentiation induction. Thereafter, the expression fetal mice at E9 and E11 days of gestation to analyze whether level of CYP3A11 increased with the maturation of the hepa- CYP2R1 and CYP26A1 were expressed at the stage of hepa- tocytes. CYP2R1 was first expressed on day 3 of differentia- toblast appearance. tion induction, and its expression was highest on day 7, after The results revealed that CYP2R1 and CYP26A1 were which it decreased with further differentiation induction. In expressed in the hepatic primordium of fetal mice at E9 days addition, high expression of CYP26A1 was observed on day of gestation and that some of the CYP2R1-positive cells and 3 of hepatocyte differentiation induction; its expression was CYP26A1-positive cells were also positive for DLK1 (Fig. 4). decreased on day 7 before being highly expressed again on Furthermore, many cells in the liver of fetal mice at E11 days day 18 (Fig. 2). Intriguingly, CYP2R1 and CYP26A1 were of gestation were positive for both CYP2R1 and CYP26A1, both induced prior to AFP and DLK1, which are makers of and a significant number of them also expressed DLK1 (Fig. hepatoblasts. 4). Expression Patterns of CYP2R1 and CYP26A1 dur- Based on these results, the probable involvement of ing the Induction of the Differentiation of ES Cells into CYP2R1 and CYP26A1 in the differentiation of hepatoblasts Hepatocytes mRNA expression of CYP2R1 and CYP26A1 was also confirmed in the process of mouse liver development. was observed prior to the differentiation of ES cells into hepa- Induction of Delta-Like 1-Positive Cells by Forced Ex- toblasts. Therefore, to evaluate whether both CYPs were also pression of CYP2R1 and CYP26A1 in ES Cells Because expressed at the protein level at that time, immunostaining the involvement of CYP2R1 and CYP26A1 in the differentia- was performed using anti-CYP2R1 Ab or anti-CYP26A1 Ab tion of hepatoblasts was indicated by the results of our above and an Ab for delta-like 1 (DLK-1), a marker of hepatoblasts. experiments, to further verify this finding, we investigated As a result, on day 3 of differentiation induction, when ES whether forced expression of CYP2R1 or CYP26A1 in ES cells had differentiated into endoderm, although CYP2R1 and cells would produce DLK1-positive cells. Expression plasmids CYP26A1 were already expressed, the expression of DLK1, a encoding CYP2R1 or CYP26A1 were transfected into ES marker of hepatoblasts, was not observed (Fig. 3). On day 7, cells, and differentiated state of the cells were examined at many cells expressing CYP2R1 or CYP26A1 were positive for 72 h after transfection. Our results showed that GFP express- DLK1 (Fig. 3). ing cells (expressing CYP2R1 or CYP26A1) differentiated Based on these results, it is possible that CYP2R1 and into hepatoblasts that were positive for DLK1 (Fig. 5). CYP26A1 are expressed prior to hepatoblast markers during Forced Expression of CYP2R1 and CYP26A1 in Mouse hepatocyte differentiation and that they play a particular roles Liver Induces DLK1-Positive Cells In the in vitro experi- in the process of differentiation of ES cells into hepatoblasts. ments, it was revealed that forced expression of CYP2R1 or Vol. 39, No. 12 (2016) Biol. Pharm. Bull. 2019

Fig. 5. Forced Expression of CYP26A1 and CYP2R1 in ES Cells Vehicle, CYP2R1 and CYP26A1 were expressed in ES cells. The cells were in- cubated for 72 h and immunostained with DLK1 Ab and anti-GFP Ab.

Fig. 3. Patterns of Expression of CYP26A1 or CYP2R1 during the In- duction 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.

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 immuno- stained with anti-EGFP and anti-DLK1 antibodies.

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 en- dodermal cells to hepatocytes are the factors secreted during Fig. 4. Patterns of Expression of CYP26A1 and CYP2R1 in the Fetal the development of hepatoblast tissues. The budding of hepa- Liver toblast tissue is initiated by stimulation by fibroblast growth Frozen sections of the liver of fetal mice at days 9 and 11 of gestation were factor (FGF) secreted by the adjacent cardiac mesoderm and made, and the expression patterns of CYP2R1 and CYP26A1 in the hepatic primor- dium were analyzed by immunohistochemical staining. 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 CYP26A1 caused differentiation of ES cells into hepato- (OsM), which are involved in the maturation of fetal hepato- blasts. Thus, in this study, we investigated whether forced cytes. ES cells were reported to be efficiently differentiated expression of CYP2R1 and CYP26A1 would differentiate into into hepatocytes by the addition of a combination of FGF-1, hepatoblasts in mouse liver. For gene transfer to the liver, FGF-4, and HGF, which are molecules that are increased dur- mice received a tail vein injection of plasmid DNA (Vehicle, ing hepatic damage, to the culture medium.11) CYP2R1, CYP26A1) dissolved in a hydrodynamic solution. This study used the differentiation induction system to dif- The results showed that GFP expressing cells differentiated ferentiate ES cells into hepatocytes to examine CYP molecu- into DLK1-positive cells (Fig. 6). lar species that contribute to the proliferation and differentia- tion of the fetal liver. In the process of differentiation induc- DISCUSSION tion from ES cells into hepatocytes, the CYP molecular spe- cies that showed interesting expression patterns were CYP2R1 To efficiently induce differentiation of ES cells into he- and CYP26A1. CYP2R1 and CYP26A1 are involved in the patocytes, it is essential to cause differentiation of ES cells metabolic pathways of vitamin A and vitamin D, respectively. 2020 Biol. Pharm. Bull. Vol. 39, No. 12 (2016)

CYP26A1 contributes to the synthesis and metabolism of the intracellular domain of E-cadherin and is known to be a retinoic acid. β-Carotene and retinyl ether are converted to regulator of gene transcription. Beta-catenin has been reported retinol (alcohol) and subsequently oxidized to retinal (alde- to be transiently expressed during liver development.19) Addi- hyde) and then retinoic acid, which has physiological action tionally, knockout of the β-catenin gene in mouse hepatoblasts after it undergoes further oxidization.15) It is known that the has been shown to decrease cellular proliferative potency, metabolism of retinoic acid involves CYP26 family mem- resulting in embryonic lethal.20) Based on these reports, the bers in the embryonic stage and CYP3A7 in the fetal stage transient expression of CYP2R1 may contribute to the pro- to regulate the concentration of retinoic acid. Retinoic acid liferation of hepatoblasts via VDR. In this study, a pattern of contributes to development and differentiation by binding to expression of CYP2R1 was observed: its expression increased nuclear receptors, such as retinoic acid receptor (RAR) and from days 3 to 7 of differentiation induction and was reduced retinoid X receptor (RXR).16) Vitamin A is the collective name thereafter. Therefore, it was suggested that CYP2R1 may play for retinal, retinol and retinoic acid and is associated with the an important role in the early process of differentiation of ES differentiation, proliferation, morphogenesis, and apoptosis of cells into hepatocytes (hepatoblasts). cells. Because an excessive intake of vitamin A also causes Transfection assay for in ES cells using an expression vec- teratogenicity, regulation of the concentration of retinoic acid tor of CYP2R1 or CYP26A1 resulted in DLK1-positive cell is highly important for the living body. In CYP26A1 knock- induction (Fig. 5); however, it remains unknown whether out mice, which show embryonic lethal or death shortly after CYP2R1 and CYP26A1 regulate the differentiation of ES birth, morphological defects are particularly observed in the cells into hepatocytes by metabolizing vitamin D and/or tailbud and hindbrain, likely because CYP26A1 expression vitamin A in the culture medium or endogenous vitamin D is localized there.7) Thus, in CYP26A1 knockout mice, the and/or vitamin A. Furthermore, in the in vivo forced expres- concentration of retinoic acid is elevated in those areas, re- sion experiments of CYP2R1 and CYP26A1, it was unclear sulting in the development of hypoplasia in the lower part of which cell types were transfected (Fig. 6). Therefore, it is the body, which generates a mermaid-like morphology. The unknown whether undifferentiated hepatic stem cells or adult results of this study showed that CYP26A1 was first expressed hepatocytes differentiated into hepatoblasts. To clarify the on day 3 of differentiation induction. Because retinoic acid mechanisms of the development and regeneration of the liver, had been added to the culture medium until day 3, it was sug- detailed analyses of the functions of CYP2R1 and CYP26A1 gested that the expression of CYP26A1 was possibly induced are necessary. to metabolize that retinoic acid to control its concentration. In this study, in both the induction of differentiation of The expression of both CYP2R1 and CYP26A1 was ob- ES cells into hepatocytes experiments and the fetal mice ex- served in the period of day 3 and 7 in this culture system, periments, the expressions of CYP2R1 and CYP26A1 were in- which is the period of differentiation from an endoderm to duced prior to the expression of DLK1, a hepatoblast marker. a hepatoblast. This day 3 and 7 period of ES cells is consid- These results suggest the involvement of these CYPs in hepa- ered to be equal to Embryonic day of 8 and 13. At the onset toblast differentiation through the metabolism of endogenous of liver development at approximately mouse embryonic day substances, such as vitamins D and A. (E9). Therefore, we could not isolate liver alone in this em- Uncovering the mechanism of CYP2R1 and CYP26A1 ex- bryonic stage (E8) and analyzed the hepatic primordium (also pression regulation will likely help clarify the mechanism of called liver bud) in E9. early liver development and/or liver regeneration. CYP26A1 was also shown to be expressed in the hepatic primordium of fetal mice at E9 to E11 days of gestation (Fig. Acknowledgments The Ministry of Education, Culture, 4). In recent years, the ability of primary hepatocytes from Sports, Science and Technology (MEXT) of Japan-Supported mice at E14.5 d of gestation to store glycogen has been re- Program for the Strategic Research Foundation at Private Uni- ported to be increased by the addition to culture medium of versities, 2014–2018, S1411019. We thank Ms. Haruka Kato, all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9CRA), Ms. Misa Iizuka, Ms. Konomi Oba, Ms. Mami Nakai, Ms. ligands of RAR and RXR, respectively.17) Based on these Saori Tomita, Ms. Tomoka Yasukawa, for their technical as- findings, CYP26A1 expression in hepatoblasts was suggested sistance. to be involved in the differentiation of endoderm cells into hepatoblasts by binding to nuclear receptors, such as RAR Conflict of Interest The authors declare no conflict of and RXR, resulting in the regulation of the concentrations of interest. ATRA and 9CRA in the living body. 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