2022 Volume 47 Issue 6 Pages 249-255
Retinoic acid, an active form of vitamin A, plays very important roles in mammalian embryogenesis. The concentration of retinoic acid is extremely low and strictly regulated by enzymes of cytochrome P450 (CYP) family, CYP26s (CYP26A1, CYP26B1 and CYP26C1) in the cells. Therefore, it is thought that changes in CYP26s activities due to exposure to a wide variety of drugs and chemicals exhibit teratogenicity. In this study, to easily detect the changes in retinoic acid level, we constructed an adenovirus-mediated reporter assay system using the promoter region of the CYP26A1 gene and inserting retinoic acid response element (RARE) and retinoid X response element (RXRE) into the downstream of the luciferase gene of reporter plasmid, which highly increased the response to retinoic acid. Reporter activity significantly increased in a concentration-dependent manner with retinoic acid; this increase was also observed at least after treatment with a very low concentration of 1 nM retinoic acid. This increase was suppressed by the accelerated metabolism of retinoic acid due to the overexpression of CYP26A1; however, this suppression was almost completely suspended by treatment with talarozole, a CYP26 inhibitor. In conclusion, the reporter assay system constructed using the induction of CYP26A1 expression is a risk assessment system that responds to extremely low concentrations of retinoic acid and is useful for assessing the excess vitamin A mediated teratogenicity caused by various chemicals at the cellular level.
Approximately 2,000 chemical substances are known to be teratogens. In particular, cases of teratogenicity caused by medications have been reported, including limb and heart malformations caused by the sleep-inducing agent, thalidomide, and achondroplasia and microcephaly caused by the anticoagulant, warfarin (Meador and Loring, 2016; Vargesson, 2015; Basu et al., 2016). Vitamin A is involved in the regulation of vision, cell differentiation and development, and the expression of genes that control skeletal patterning. It has been reported that excessive intake of vitamin A by pregnant women during the skeletal development of the fetus can result in bone malformations in the newborn (Schaefer et al., 2010; Rhinn and Dollé, 2012; Uehara et al., 2009).
Vitamin A is a general term for fat-soluble retinoids such as retinol, retinal, and retinoic acid. The retinoic acid biosynthetic pathway is initiated by the conversion of retinol to retinal aldehyde by retinol dehydrogenases (RDHs). Retinal aldehyde is then converted by retinal dehydrogenases (RALDHs) into all-trans-retinoic acid (atRA), which is interchangeable with 9-cis retinoic acid (9-cisRA). It is also known that atRA is metabolized by CYP26s, and the intracellular level of atRA are thought to be regulated by the balance between RALDHs and CYP26s activities (Strate et al., 2009). atRA and 9-cisRA are agonists of retinoic acid receptor (RAR) and promote the formation of heterodimers with the nuclear receptors RAR and retinoid X receptor (RXR) and homodimers of the RAR, and 9-cisRA functions as an agonist of both RAR and RXR receptors (Allenby et al., 1993; Repa et al., 1993). These complexes activate transcription by binding to the retinoic acid response element (RARE) or retinoid X response element (RXRE) in the promoter region of CYP26s (Loudig et al., 2000).
Vitamin A deficiency has been reported to cause undifferentiated fetal malformations, such as monocular disease. The level of vitamin A is one of the most important factors for normal fetal development (Abu-Abed et al., 2001). However, the level of vitamin A in the fetus is extremely low and it is very difficult to quantitatively analyze its level in experimental animals using conventional measurement methods, such as high-performance liquid chromatography (Kochhar et al., 1988). Therefore, in this study, we constructed a reporter assay system using the promoter region of CYP26A1, which is strongly expressed by atRA, and examined its usefulness at the cellular level.
HepG2, HeLa and MCF-7 cells, were obtained from the Cell Bank of the Riken Bioresource Research Center (Tsukuba, Japan). Those cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Biowest, Miami, FL, USA), 1 × antibiotic-antimycotic (100 U/mL penicillin G sodium,100 µg/mL streptomycin sulfate, 0.25 µg/mL amphotericin B) (Invitrogen, Carlsbad, CA, USA), 1 × MEM non-essential amino acid solution (Invitrogen), 0.45% glucose, and 2 mM L-glutamine (Wako), and incubated at 37°C in a humidified atmosphere of 95% air/5% CO2.
Preparation of reporter plasmidsThe pGL4.14 [luc2/Hygro] vector and human CYP26A1 cDNA cloned into the Gateway Entry vector, pENTR223.1 26A1 cDNA were purchased from Promega and Thermo Fisher Scientific (Waltham, MA, USA), respectively. The enhancer/promoter region of CYP26A1 gene (approximately 3.8 kbp) shown in Fig. 1A as CYP26A1 promoter was amplified from a human genomic DNA using KOD-Plus-Neo (Toyobo, Osaka, Japan). The following primers were used: CYP26A1 (sense) 5′-gaggctagcGCTGCCCAGACTTCTCTCTGAGTATAACC-3′ and CYP26A1 (anti-sense) 5′-gagctcgagAAGTCCTTTCAGGAATCGTCCAGCTGCCT-3′. The amplified PCR product was incorporated into the pCR-XL-TOPO® vector (Invitrogen) by using a TOPO® cloning reaction. To obtain the pGL4.26A1 reporter plasmid, the subcloned CYP26A1 enhancer/promoter DNA was isolated with NheI and XhoI (New England Biolabs, Ipswich, MA, USA) and further subcloned into NheI and XhoI sites of the pGL4.10 [luc2 Hygro] vector. The pGL4.26A1/RARE reporter plasmid (pGL4.26A1/RARErp) was made by inserting the RARE/RXRE responsive element (26 bp) into BamHI and SalI sites of pGL4.26A1 reporter plasmid. The RARE/RXRE DNA element was made by annealing with RARE/RXRE (sense) 5′-ggcgtcgacGGGTAGGGGTCACGAAAGGTCACTCGggatccgcc-3′ and RARE/RXRE (anti-sense) 5′-ggcggatccCGAGTGACCTTTCGTGACCCCTACCCgtcgacgcc-3′ (Mader et al., 1993). The inserted DNA fragment was confirmed by sequencing.
Effects of retinoic acid response element (RARE) on the reporter activity through the enhancer/promoter region of the CYP26A1 gene. A. Construction of two types of reporter plasmids, a) pGL4. 26A1 and b) pGL4. 26A1/RARErp. B. MCF-7 cells were seeded onto 48-well tissue culture plates at 3 × 104 cells/well. After 24 hr for incubation, cells were transfected with each reporter plasmid. After a further 24 hr, the cells were treated with all-trans-retinoic acid (atRA) for 24 hr. The reporter activity was measured using Dual-Luciferase assay system. Data represent the mean ± SD (n = 3); Dunnett’s test, *p < 0.05 (vs each control group).
The cells were transfected by using the lipofection method using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.
Production of CYP26A1-expressing Adenovirus (Ad-CYP26A1)The synthesis of Ad-CYP26A1 plasmid was carried out according to the manufacturer’s protocol for the ViraPowerTM Adenoviral Expression System, pENTR223.1 26A1 cDNA and pAd/CMV/V5-DESTTM Gateway® vector kits (Invitrogen). The plasmid was purified by CsCl density gradient centrifugation, linearized with PacI (New England Biolabs) and then transfected into 293T cells using Targefect F-1 (Nacalai Tesque, Kyoto, Japan). The control adenovirus, β-galactosidase-expressing adenovirus (Ad-LacZ) was produced using the pAd/CMV/V5-GW/lacZ control plasmid (Invitrogen). The titer of adenovirus, 50% titer culture infectious dose (TCID50), was determined in 293T cells. TCID50 has been reported almost equivalent to that of the plaque-forming unit (Sasaki et al., 2013). Multiplicity of infection (MOI) was calculated by dividing TCID50 by the number of cells at the time of seeding. The expression of recombinant CYP26A1 was confirmed by Western blotting.
Production of Ad-CYP26A1/RARErpThe DNA fragment form the CYP26A1 enhancer/promoter DNA (-3798 bp to -40 bp) to the RARE/RXRE responsive element including the luc2 reporter gene (approximately 2 kbp) of the pGL4.26A1/RARErp was amplified by KOD-Plus-Neo (Toyobo) using following primers, pGL4.10 [luc2 Hygro] vector; (sense) 5′-caccATTTCTCTGGCCTAACTGGCC-3′ and (anti-sense) 5′-TGACTGGGTTGAAGGCTCAAGGG-3′. The amplified product was cloned into a pENTR/SD/D-TOPO vector (Thermo Fisher Scientific). To confirm the insert (approximately 5.8 kbp), the pENTR-CYP26A1/RARErp TOPO vector was treated with restriction enzymes AscI and NotI (New England Biolabs). pENTR-CYP26A1/RARErp TOPO vector was replaced with the pAd/CMV/V5-DEST gateway vector (Thermo Fisher Scientific) using GATEWAY®LR ClonaseTM II enzyme mix (Invitrogen). Ad-CYP26A1/RARErp was made according to previously described in Ad-CYP26A1.
Adenovirus infection of cells and reagent treatmentMCF-7 cells were seeded at 1.5 × 104 cells/well in 48-well plates and cultured for 48 hr. Then, 100 µL/well of virus solution [adjusted to each multiplicity of infection (MOI) in DMEM using Ad-LacZ, Ad-CYP26A1 and Ad-CYP26A1/RARErp (1.0 × 108 PFU/mL)] was added, and the cells were cultured for 1 hr to become infected. After infection, 200 µL of DMEM was added, the cells were incubated for 24 hr, treated with atRA (Wako) and/or talarozole (Wako), and assayed after another 24 hr.
Luciferase AssayLuciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The medium in each well of the reagent-treated cells cultured in 48-well plates was removed, and the cells were washed twice with an equal volume of sterile phosphate-buffered saline (PBS; 300 µL/well). Next, 70 µL of passive lysis buffer diluted 5-fold with sterile purified water was added to each well, and the wells were shaken for 10 min at room temperature. The cells were then suspended by pipetting and the resulting cell lysate was transferred to a new tube. The tube was centrifuged at 400 × g for 10 min at 4°C, and 25 µL of the supernatant was used as the sample and transferred to a 96-well white plate. To simultaneously measure firefly luciferase and Renilla luciferase activities, 25 µL of Luciferase Assay substrate (Promega) was added to each well, followed by 25 µL of Stop&Glo substrate diluted 50-fold with the Stop&Glo reagent. A GlomaxTM 96 microplate luminometer (Promega) was used for measurements. In addition, 10 µL of the supernatant was transferred to another 96-well plate, and 200 µL of a solution of Bio-Rad protein assay reagent solution (Bio-Rad Laboratories, Hercules, CA, USA) diluted 5-fold with PBS was added and shaken for 10 min. Absorbance at 595 nm was measured using a POWERSCAN® HT microplate reader (BioTek, Winooski, VT, USA). Protein concentration was determined using the Bradford method, and protein correction was performed based on the results of the Luciferase reporter assay.
Statistical analysesOne-way ANOVA with Dunnett’s test was performed using StatView 5.0 software. A Student’s t-test for Microsoft Excel was used to compare the two groups. Each experiment shown in figures was carried out at least three times.
At first, when pGL4.26A1 was transfected to HepG2, HeLa and MCF-7 cells, induction fold of the reporter activity by atRA was the highest in MCF-7 cells (data not shown). Therefore, we chose MCF-7 cells for this experiment. To further obtained higher induction fold, an extra RARE/RXRE responsive element was added to pGL4. 26A1 plasmid (pGL4.26A1/RARErp, Fig. 1A). Both reporter activities derived from pGL4. 26A1 and pGL4.26A1/RARErp were compared in Fig. 1B. When cells were treated with atRA up to a final concentration of 10 nM, the reporter activity was higher in cells transfected with pGL4.26A1/RARErp which have the addition of the RARE/RXRE binding element downstream of the luciferase gene.
Next, we generated an adenovirus (Ad-CYP26A1/RARErp) from CYP26A1/RARErp plasmid. Cells were infected with adenovirus at an MOI of 50 and treated with atRA at final concentrations ranging from 0.1 nM–10 µM. As a result, the reporter activity was significantly increased in a concentration-dependent manner up to 1 µM, and approximately 4-fold induction was observed even at a low concentration of 1 nM (Fig. 2).
Effects of retinoic acid on reporter activity in the cells infected with Ad-CYP26A1/RARErp. MCF-7 cells were seeded onto 48-well tissue culture plates at 1.5 × 104 cells/well. After 48 hr of incubation, the cells were infected with Ad-CYP26A1/RARErp adenovirus at an MOI of 50. After a further 24 hr, the cells were treated with various concentrations of atRA for 24 hr. The reporter activity was measured using a Luciferase assay system. Data represent the mean ± SD (n = 3); Dunnett’s test, *p < 0.05, **p < 0.01 (vs each control group).
The CYP26 family includes three molecular species, of which CYP26A1 is mainly involved in retinoic acid metabolism (Abu-Abed et al., 2001). Then the effect of CYP26A1 overexpression was examined on the increase in reporter activity of Ad-CYP26A1/RARErp. As result, the increase in reporter activity induced by atRA was significantly suppressed in response to the degree of CYP26A1 overexpression (Fig. 3). This suggests that the concentration of atRA in cells was reduced due to the accelerated metabolism of atRA by CYP26A1 overexpression. It was also observed that overexpression of CYP26A1 reduced the reporter activity of the control. This reduction was an MOI dependent manner, suggesting that the medium used in the experiment may have contained undetectable trace amounts of retinoic acid. It is also possible that the expression of Ad-CYP26A1/RARErp may be reduced when used in combination with other adenovirus vectors due to sharing of intracellular transcription factors. However, the details are currently unknown.
Effects of co-infection of Ad-CYP26A1/RARErp and Ad-CYP26A1 on reporter activity. MCF-7 cells were seeded 48-well tissue culture plates at 1.5 × 104 cells/well. After 48 hr of incubation, cells were co-infected with Ad-CYP26A1/RARErp adenovirus at an MOI of 50, and Ad-CYP26A1 adenovirus at an MOI of 10 or 100. Adenovirus carrying the LacZ gene (Ad-LacZ) was used as a control (10 MOI). After 24 hr, the cells were treated with various concentrations of atRA for 24 hr. The reporter activity was measured using a Luciferase assay system. Data represent the mean ± SD (n = 3); Dunnett’s test, **p < 0.01 (vs each control group); ##p < 0.01 (vs Ad-LacZ).
We next examined the effect of talarozole, a CYP26 inhibitor, on retinoic acid metabolism (Stoppie et al., 2000). The reporter activity of Ad-CYP26A1/RARErp, which was increased by atRA, was significantly inhibited by CYP26A1 overexpression, but showed an upward trend from 1 nM tararozole treatment; no inhibitory effect of CYP26A1 overexpression was observed at 3.3 nM (Fig. 4) under 10 MOI infection of Ad-CYP26A1. Another CYP26 inhibitor, a ketoconazole, also suspended the decrease in reporter activity due to CYP26A1 overexpression (data not shown). These results suggest that the reporter activity is mainly dependent on the level of atRA in cells metabolized by CYP26A1.
Effects of talarozole on the reporter activity in the cells co-infected with Ad-CYP26A1/RARErp and Ad-CYP26A1. MCF-7 cells were seeded onto 48-well tissue culture plates at 1.5 × 104 cells/well. After 48 hr of incubation, the cells were co-infected with Ad-CYP26A1/RARErp adenovirus at 50 MOI and Ad-CYP26A1 adenovirus at 10 MOI. Adenovirus carrying the LacZ gene (Ad-LacZ) was used as a control (10 MOI). After 24 hr, the cells were treated with atRA (10 nM), and talarozole (1 or 3.3 nM) for 24 hr. The reporter activity was measured using a Luciferase assay system. Data represent the mean ± SD (n = 3); Dunnett’s test **p < 0.01 (vs each control group), t-test ##p < 0.01.
In this study, we developed an adenovirus-mediated reporter assay system by use of enhancer/promoter region of CYP26A1 gene and by adding RARE/RXRE responsive element. Mader et al. reported that atRA forms a RAR/RAR homodimer and that this complex binds strongly to the DR4G sequence of the PuGGTCA motif among several reported element sequences (Mader et al., 1993; Al Tanoury et al., 2013). In the current study, an addition of the RARE/RXRE responsive element, to which the homodimers and heterodimers of RAR or RXR bind, also increased the response to atRA. This system responds to atRA by indirectly monitoring the reporter activity, and the response to atRA was also enhanced a concentration-dependent manner. In this reporter assay, the promoter activity of Ad-CYP26A1/RARErp was shown to be responsive to atRA at a final concentration of 1 nM to 1 µM, with an approximately 4-fold increase in activity even at a low concentration of 1 nM.
It has been reported that the measurable retinoic acid concentration in fetal organs is a few nanomole at the highest estimate, and the application of our evaluation system constructed in this study may lead to the quantification of retinoic acid concentration in fetal organs (Kochhar et al., 1988). In this experiment, the reporter activity induced by 1 nM atRA was strongly reduced by overexpression of CYP261. atRA was primarily metabolized to hydroxylated forms (4-OH-atRA and 18-OH-atRA) by CYP26A1; however 4-OH-atRA has still lower affinity for RARs than that of atRA (Topletz et al., 2015).
Humans are constantly exposed to chemical substances such as pesticides and food additives, and some of these substances are teratogenic (Kalliora et al., 2018). The adenovirus-mediated reporter assay system constructed in this study is useful for monitoring teratogenicity caused by chemical substances through changes in the cell level of vitamin A. Furthermore, the development of experimental animals using this assay system is expected to make a significant contribution to the evaluation of teratogenicity of chemical substances in in vivo studies.
This work was supported by a grant from the Food Safety Commission, Cabinet Office, Government of Japan (Research Program for Risk Assessment Study on Food Safety, No. 1402). We would like to thank Editage (www.editage.com) for English language editing.
Conflict of interestThe authors declare that there is no conflict of interest.
